Several genetic variants in the mitochondrial genome (mtDNA), including ancient polymorphisms, are associated with chronic inflammatory conditions, but investigating the functional consequences of such mtDNA polymorphisms in humans is challenging due to the influence of many other polymorphisms in both mtDNA and the nuclear genome (nDNA). Here, using the conplastic mouse strain B6-mtFVB, we show that in mice, a maternally inherited natural mutation (m.7778G > T) in the mitochondrially encoded gene ATP synthase 8 (mt-Atp8) of complex V impacts on the cellular metabolic profile and effector functions of CD4+ T cells and induces mild changes in oxidative phosphorylation (OXPHOS) complex activities. These changes culminated in significantly lower disease susceptibility in two models of inflammatory skin disease. Our findings provide experimental evidence that a natural variation in mtDNA influences chronic inflammatory conditions through alterations in cellular metabolism and the systemic metabolic profile without causing major dysfunction in the OXPHOS system.

Bullous pemphigoid (BP) is the most common of pemphigoid diseases caused by autoantibodies against the structures of dermoepidermal junction followed by complement activation, innate immune cell infiltration, neutrophil proteinase secretion and subepidermal blister formation. The first-line treatment of BP is topical and systemic glucocorticoids (GC). Regulation of the immune system and inflammatory cells is the main target of GC actions. GCs act through genomic and non-genomic mechanisms. The human glucocorticoid receptor (GR) mediates most of the biologic effects of GC: cytosolic GR binds GCs and is capable to bind to glucocorticoid response elements in DNA and either transactivate or transrepress genes depending on the tissue and cell type. In addition, GR exerts rapid, non-genomic effects possibly mediated by membrane-localized receptors or by translocation to mitochondria. GCs can also interact directly with several enzymes and cytokines. As a target treatment for BP, the production of autoantibodies should be discontinued. GCs, in spite of their wide immunosuppressive actions, are weak to stop immunoglobulin G (IgG) autoantibody formation. However, both systemic and topical GCs are able to reduce the clinical symptoms of BP. GCs are used to inhibit the secondary inflammation and symptoms, such as blistering and pruritus, and it is shown that GC treatment will gradually decrease also the autoantibody formation. Our review article analyses the mode of action of GC treatment in BP, as far it is possible due to paucity of modern immunological studies.

Neutrophil granulocytes are the first leukocytes that encounter and phagocytose Leishmania major (L. major) parasites in the infected skin. The parasites can nonetheless survive within neutrophils. However, the mechanisms enabling the survival of Leishmania within neutrophils are still elusive. Previous findings indicated that human neutrophils can engulf apoptotic cells. Since apoptotic neutrophils are abundant in infected tissues, we hypothesized that the uptake of apoptotic cells results in diminished anti-leishmanial activity and, consequently, contributes to enhanced survival of the parasites at the site of infection. In the present study, we demonstrated that L. major-infected primary human neutrophils acquire enhanced capacity to engulf apoptotic cells. This was associated with increased expression of the complement receptors 1 and 3 involved in phagocytosis of apoptotic cells. Next, we showed that ingestion of apoptotic cells affects neutrophil antimicrobial functions. We observed that phagocytosis of apoptotic cells by neutrophils downregulates the phosphorylation of p38 MAPK and PKCδ, the kinases involved in activation of NADPH oxidase and hence reactive oxygen species (ROS) production. In line, uptake of apoptotic cells inhibits TNF- and L. major-induced ROS production by neutrophils. Importantly, we found that the survival of Leishmania in neutrophils is strongly enhanced in neutrophils exposed to apoptotic cells. Together, our findings reveal that apoptotic cells promote L. major survival within neutrophils by downregulating critical antimicrobial functions. This suggests that the induction of enhanced uptake of apoptotic cells represents a novel evasion mechanism of the parasites that facilitates their survival in neutrophil granulocytes.

Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases.

A broad range of immunosuppressive and immunomodulatory effects of heat shock protein 90 (Hsp90) blockade has been described in models of autoimmune bullous diseases, but the direct contribution of this chaperone to neutrophil effector pathways in the context of autoantibody-driven blistering is generally unknown. Therefore, this has been addressed in the current study on the basis of the subepidermal blistering disease epidermolysis bullosa acquisita (EBA) characterized by autoantibodies against type VII collagen, in which a crucial role of neutrophils and both their reactive oxygen species and matrix metalloproteinases in mediating tissue injury has been established. First, the Hsp90 antagonist 17-DMAG dose-dependently inhibited dermal-epidermal separation ex vivo in cryosections of human skin induced by co-incubation of EBA patient autoantibodies with neutrophils from healthy blood donors. Next, 17-DMAG dose-dependently suppressed production and release of reactive oxygen species by human neutrophils induced by both fMLP ± LPS and EBA-specific immune complexes. In addition, co-immunoprecipitation studies revealed that extracellular Hsp90 interacted with secreted matrix metalloproteinases 2 and 12 in sera of EBA patients, suggesting that these basement membrane-degrading proteolytic enzymes are client proteins of Hsp90 and dependent on its chaperone function. Our findings add to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade and implicate that Hsp90 is closely involved in the effector mechanisms of neutrophil-driven autoantibody-induced tissue damage, thus being a relevant therapeutic target in patients with neutrophil-mediated autoimmune diseases such as inflammatory types of EBA.

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MP's therapeutic effects.

Neutrophil granulocytes provide the first line of defense against bacterial, fungal, and parasitic infections. They phagocytose and kill many invading pathogens. Certain pathogenic microorganisms such as the intracellular protozoan parasite Leishmania major ( L. major ) can survive inside neutrophils. Mature neutrophils have a very short life span due to spontaneous apoptosis. Previously, we have reported that infections with L. major are able to delay spontaneous apoptosis. In the present study, we addressed the underlying mechanisms of regulation of both extrinsic and intrinsic apoptosis. We show that interaction with L. major transiently activates ERK1/2 phosphorylation. Pharmacological inhibition of ERK1/2 phosphorylation reversed the apoptosis delay. Moreover, infection leads to the enhanced and sustainable expression of the anti-apoptotic proteins Bcl-2 and Bfl-1, respectively. As downstream events, the release of cytochrome c from mitochondria and processing of caspase-6 were inhibited. We also confirm that infection with L. major results in reduced FAS expression on the surface of neutrophils. The presented data indicate that infection with L. major affects both intrinsic as well as extrinsic pathways of neutrophil apoptosis. Enhanced life span of host neutrophils enables the parasite to survive within neutrophils.

ABSTRACT Anaplasma phagocytophilum , a Gram-negative, obligate intracellular bacterium infects primarily neutrophil granulocytes. Infection with A. phagocytophilum leads to inhibition of neutrophil apoptosis and consequently contributes to the longevity of the host cells. Previous studies demonstrated that the infection inhibits the executionary apoptotic machinery in neutrophils. However, little attempt has been made to explore which survival signals are modulated by the pathogen. The aim of the present study was to clarify whether the phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB signaling pathways, which are considered as important survival pathways in neutrophils, are involved in A. phagocytophilum -induced apoptosis delay. Our data show that infection of neutrophils with A. phagocytophilum activates the PI3K/Akt pathway and suggest that this pathway, which in turn maintains the expression of the antiapoptotic protein Mcl-1, contributes to the infection-induced apoptosis delay. In addition, the PI3K/Akt pathway is involved in the activation of NF-κB in A. phagocytophilum -infected neutrophils. Activation of NF-κB leads to the release of interleukin-8 (IL-8) from infected neutrophils, which, in an autocrine manner, delays neutrophil apoptosis. In addition, enhanced expression of the antiapoptotic protein cIAP2 was observed in A. phagocytophilum -infected neutrophils. Taken together, the data indicate that upstream of the apoptotic cascade, signaling via the PI3K/Akt pathway plays a major role for apoptosis delay in A. phagocytophilum -infected neutrophils.

Autoantibody‐mediated diseases are clinically heterogeneous and often fail conventional therapeutic strategies. Gene expression profiling has helped to identify new molecular pathways in these diseases, although their potential as treatment targets largely remains to be functionally validated. Based on weighted gene co‐expression network analysis, we determined the transcriptional network in experimental epidermolysis bullosa acquisita (EBA), a paradigm of an antibody‐mediated organ‐specific autoimmune disease characterized by autoantibodies directed against type VII collagen. We identified 33 distinct and differentially expressed modules, including Fcγ receptor (FcγR) IV and components of the neutrophil‐associated enzyme system in autoantibody transfer‐induced EBA. Validation experiments, including functional analysis, demonstrated that FcγRIV expression on neutrophils crucially contributes to autoantibody‐induced tissue injury in the transfer model of EBA. Mice lacking the common γ‐chain of activating FcγRs, deficient in FcγRIV or treated with FcγRIV function blocking antibody, but not mice deficient in FcγRI, FcγRIIB, FcγRIII or both FcγRI and FcγRIII, were effectively protected from EBA. Skin disease was restored in γ‐chain‐deficient mice locally reconstituted with neutrophils from wild‐type, but not from γ‐chain‐deficient, mice. Our findings both genetically and functionally identify a novel disease‐related molecule, FcγRIV, in an autoantibody‐mediated disorder, which may be of importance for the development of novel targeted therapies. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recently, we have reported that, in addition to macrophages, also neutrophil granulocytes can phagocytose apoptotic neutrophils. Based on this finding, we hypothesized that “cannibalistic” neutrophils at sites of acute infection/inflammation play a major role in the clearance of apoptotic neutrophils. Since at sites of infection/inflammation neutrophils are exposed to microbial constituents and proinflammatory cytokines, in the present study we analyzed the effect of TLR-ligands and cytokines on the ability of neutrophils to phagocytose apoptotic cells in vitro . We observed that exposure to ligands of TLR2 (Malp2, Pam3CSK4), TLR4 (LPS), TLR7/TLR8 (R848), and TLR9 (ODN 2006) led to increased phagocytosis of apoptotic cells by neutrophils. In addition, proinflammatory cytokines such as TNF and GM-CSF strongly enhanced the uptake of apoptotic cells by neutrophils. These results support the hypothesis that neutrophils acquire the ability to phagocytose apoptotic cells at sites of acute infection/inflammation and thereby can contribute to the resolution of inflammation.

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-γ results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-α and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-α, which was independent of NF-κB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

The vascular addressins mucosal addressin cell adhesion molecule-1, P-selectin and ICAM-1 permit alpha(4)beta(7)-integrin-expressing DC, especially those of the myeloid lineage (CD11c(+)CD11b(+) DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule-1 in P-selectin(-/-) mice or experimental approaches with beta 7-integrin(-/-) or ICAM-1(-/-) mice revealed that limited access or absence of CD11c(+)CD11b(+) DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into beta 7-integrin(-/-) mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c(+)CD11b(+) DC are characterized by decreased IL-15 and IL-12 mRNA and/or protein levels. Chronic administration of IL-15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL-12 positively affects the IFN-gamma expression of uNK cells. Real-time RT-PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL-15 and IL-12 in the pregnant mouse uterus.