Docosahexaenoic Acid Modulates NK Cell Effects on Neutrophils and Their Crosstalk.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadNatural killer (NK) cells and neutrophils engage in crosstalk that is important in inflammation and likely also for resolution of inflammation. NK cells activate neutrophils and induce their infiltration to the inflamed sites but may also influence their apoptosis and their subsequent efferocytosis by macrophages. Several studies indicate that docosahexaenoic acid (DHA) can inhibit NK cell cytotoxicity but the effects of DHA on the ability of NK cells to engage in crosstalk with neutrophils and affect their functions have not been described. This study explored the kinetics of the effects of NK cells and NK cells pre-treated with DHA on neutrophil surface molecule expression and apoptosis, as well as the ability of NK cells to affect other neutrophil functions. In addition, the study explored the effects of neutrophils on NK cell phenotype and function. Primary NK cells were pre-incubated with or without DHA, then stimulated and co-cultured with freshly isolated neutrophils. When co-cultured with NK cells, neutrophils had higher expression levels of CD11b and CD47; secreted more IL-8, IL-1ra, and CXCL10; had increased phagocytic ability; and their apoptosis was increased early after initiation of the co-culture while dampened at a later time-point. Pre-incubation of NK cells with DHA attenuated NK cell-induced upregulation of CD11b and CD47 on neutrophils, had minor effects on NK cell induction of cytokine/chemokine secretion or their phagocytic ability. Neutrophils also affected the function of NK cells, lowering the frequency of NKp46+ and CXCR3+ NK cells and increasing the concentrations of IFN-γ, TNF-α, and GM-CSF in the co-cultures. Pre-incubation of NK cells with DHA further decreased the frequency of NKp46+ NK cells in the co-culture with neutrophils and decreased the concentrations of IFN-γ, CCL3 and GM-CSF. These findings indicate that NK cells have mostly pro-inflammatory effects on neutrophils and that DHA can attenuate some of these pro-inflammatory effects. Neutrophils had both anti- and pro-inflammatory effects on NK cells. When NK cells had been pre-treated with DHA, the anti-inflammatory effects were increased and some of the pro-inflammatory effects attenuated. Overall, the results suggest that DHA may lead to a more anti-inflammatory microenvironment for NK cell and neutrophil crosstalk.Icelandic Research Fund University of Iceland Research Fund Landspitali University Hospital Research Fund Memorial Fund of Helga Jonsdottir and Sigurlidi Kristjansso

Type I interferon-activated microglia are critical for neuromyelitis optica pathology

Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system (CNS) most frequently mediated by serum autoantibodies against the water channel aquaporin 4, expressed on CNS astrocytes, resulting in primary astrocytopathy. There is no cure for NMO, and treatment with Type I interferon (IFNI)-IFN beta is ineffective or even detrimental. We have previously shown that both NMO lesions and associated microglial activation were reduced in mice lacking the receptor for IFN beta. However, the role of microglia in NMO is not well understood. In this study, we clarify the pathomechanism for IFNI dependence of and the role of microglia in experimental NMO. Transcriptome analysis showed a strong IFNI footprint in affected CNS tissue as well as in microglial subpopulations. Treatment with IFN beta led to exacerbated pathology and further microglial activation as evidenced by expansion of a CD11c(+) subset of microglia. Importantly, depletion of microglia led to suppression of pathology and decrease of IFNI signature genes. Our data show a pro-pathologic role for IFNI-activated microglia in NMO and open new perspectives for microglia-targeted therapies

CSF1R Stimulation Promotes Increased Neuroprotection by CD11c+ Microglia in EAE

Microglia are resident immune cells of the central nervous system. Their development and maintenance depend on stimulation of Colony Stimulating Factor-1 receptor (CSF1R). Microglia play an important role in neurodevelopment and a population of microglia that expresses the complement receptor CD11c is critical for primary myelination. This population is virtually absent in the healthy adult brain but increases dramatically upon neuroinflammatory conditions, and these microglia are suggested to play a protective role in central nervous system (CNS) diseases. To date, the molecular trigger for their expansion is unknown. Here we showed that stimulation of CSF1R by either of its ligands, CSF1 and interleukin (IL)-34, can induce expansion of CD11c+ microglia. In addition, such stimulation resulted in amelioration of EAE symptoms and decreased demyelination. Treatment with CSF1R ligands also induced expression of the chemokine CCL2, and we showed that experimental overexpression of CCL2 in the brain led to a dramatic increase of CD11c+ microglia, independent of CCR2. Moreover, this led to elevated CSF1 expression, suggesting a positive feedback loop between CSF1R and CCL2. These data provide new insights to microglia biology and open new perspectives for modulating microglial activity in neuroinflammatory diseases such as multiple sclerosis

Omega-3 polyunsaturated fatty acids promote inflammation resolution and affect natural killer cells

Acute inflammation can progress into chronic inflammation when its resolution is impaired. Chronic inflammation is estimated to contribute to up to half of all disease-associated deaths worldwide. Resolution of inflammation is a tightly regulated process characterized by ten distinct cellular and molecular hallmarks. Lipid-derived specialized pro-resolving mediators (SPMs) are a group compounds derived from omega-3 or omega-6 polyunsaturated fatty acids (PUFAs) that effectively promote inflammation resolution. Thus, it is speculated that an imbalanced consumption of omega-6 and omega-3 PUFAs can contribute to the development of chronic inflammation. Research has shown that dietary omega-3 PUFAs can promote resolution of inflammation in murine inflammatory models. Additionally, natural killer (NK) cells have been described as potential effector cells in resolving inflammation. However, the effects of omega-3 PUFAs on NK cell recruitment and on NK cell resolution effector functions is still being investigated. We aimed to determine the effects of omega-3 PUFAs on inflammation resolution and NK cell recruitment, function, and interactions with neutrophils. To investigate this, we used an antigen-induced peritonitis model and enhanced its resolution using dietary fish oil rich in omega-3 PUFAs. We also evaluated the numbers and phenotype of the NK cells accumulated in the inflamed peritoneum. We then assessed the effects of omega-3 PUFAs on human NK cell crosstalk with neutrophils and their production of oxygenized lipids. Female C57Bl/6 mice were fed a Westernized diet (control, C) enriched with 2.8% menhaden fish oil (Fo) for a total of 5 weeks. They were immunized twice with methylated bovine serum albumin (mBSA) with a two-week interval and subsequently injected intraperitoneally with mBSA to induce inflammation. Mice were sacrificed prior to and 1.5, 3, 6, and 12 h after inflammation induction and peritoneal exudate and lymph nodes collected. Human NK cells were pre-incubated with PUFAs and cultured alone or with freshly isolated neutrophils. Neutrophil and NK cell numbers and their expression of surface molecules were analyzed by flow cytometry; pro-inflammatory and pro-resolving mediator concentration by ELISA, Luminex, and LC-MS/MS; lipoxygenase expression by SimpleWestern; and apoptotic cell numbers in lymph nodes by TUNEL staining. We found that dietary fish oil can decrease peritoneal neutrophil numbers and their expression of the ‘eat-me-not’ molecule CD47, while increasing their apoptosis levels. We also detected dampened concentrations of the pro-inflammatory chemokines and cytokines; CXCL1, -2, CCL20, TNF-α, IL-6, and IL-6Rα, and omega-6 PUFA-derived eicosanoids; prostaglandins (PGs) and thromboxane (Tx)B2 in inflamed mice fed the Fo diet compared to that in mice fed the C diet. Concurrently, dietary fish oil enhanced peritoneal concentrations of insulin-like growth factor-1, transforming growth factor-β1, and soluble TNF receptor II during inflammation. Inflamed mice fed the Fo diet had higher peritoneal omega-3 to omega-6 ratio in peritoneal exudate throughout acute inflammation compared to that in mice fed the C diet. Dietary Fo promoted accumulation of CD11b+CD27- NK cells in the peritoneum at the peak of inflammation and increased peritoneal concentrations of CCL5, CXCL10, and CXCL12. The accumulated peritoneal NK cells were also found to express higher levels of CCR5 in inflamed mice fed the Fo diet compared to those fed the C diet. Interestingly, dietary fish oil increased percentages, numbers, and expression levels of peritoneal CD107a+ NK cells in inflamed mice, while the peritoneal percentages, numbers, and expression levels of CD62L+ NK cells were lowered. We found that the omega-3 PUFA, docosahexaenoic acid (DHA), attenuated human NK cell induction of CD11b and CD47 expression on neutrophils. Specifically, NK cells pre-incubated with DHA did not induce the CD11b+CD47high neutrophil subset as seen when untreated NK cells and neutrophils were cultured together. DHA also dampened NK cell-induced neutrophil apoptosis after extended co-culture of the two cell types. Interestingly, NK cells pre-incubated with DHA expressed lower levels of NKp46 than untreated NK cells when cultured with neutrophils. Finally, DHA tempered neutrophil-induced production of pro-inflammatory cytokines by NK cells. Interestingly, we found that unstimulated NK cells expressed 5-, 12-, and 15-lipoxygenases required for the synthesis of lipid-derived SPMs. Further, distinct oxylipidomic profiles were observed in NK cells cultured with arachidonic acid (AA), eicosapentaenoic acid, or DHA. In fact, NK cells cultured with omega-3 PUFAs produced both fully formed SPMs and their intermediates regardless of the presence of neutrophils. Finally, we found that NK cell cultured with AA only contained PGs or TXs if neutrophils were present. In this thesis we show that dietary fish oil enhances several hallmarks of inflammation resolution, including neutrophil apoptosis and efferocytosis. This effect may be mediated through enhanced peritoneal accumulation of cytotoxic CD11b+CD27- NK cells. In addition, the data illustrate that DHA modifies NK cell effects on and crosstalk with neutrophils. Finally, we show that NK cells express lipoxygenases and synthesize lipid-derived SPMs that promote resolution of inflammation.Bráð bólga getur þróast yfir í langvinna bólgu ef bólguhjöðnun er ábótavant. Langvarandi bólga er talin eiga þátt í allt að helmingi dauðsfalla vegna sjúkdóma í heiminum. Bólguhjöðnun er ferli sem er vel stjórnað og einkennist af tíu aðgreindum kennimerkjum sem taka til bæði frumna og sameinda. Lípíðafleidd bólguhjöðnunarboðefni er hópur sameinda sem er upprunnin frá ómega-3 eða ómega-6 fjölómettuðum fitusýrum (FÓFS) og hvetja bólguhjöðnun. Því er talið að ójafnvægi í neyslu á ómega-6 og ómega-3 FÓFS geti verið orsakaþáttur í þróun langvarandi bólgu. Niðurstöður rannsókna sýna að ómega-3 FÓFS í fæði geta ýtt undir bólguhjöðnun í tilraunadýrum. Auk þess hefur verið sýnt fram á að náttúrulegar drápsfrumur (NK frumur) taka virkan þátt í bólguhjöðnun. Þó er lítið vitað um áhrif ómega-3 FÓFS á íferð NK frumna á bólgustað og hvernig þær geta miðlað bólguhjöðnun. Markmið verkefnisins var að kanna áhrif ómega-3 FÓFS á bólguhjöðnun, íferð NK frumna á bólgustað, virkni þeirra og samspil við daufkyrninga. Til að rannsaka þetta var framkölluð vakamiðluð kviðarholsbólga í músum og bólguhjöðnun efld með því að gefa músunum fóður með ómega-3 FÓFS. Fjöldi og svipgerð NK frumna í kviðarholi var metin. Jafnframt voru áhrif ómega-3 FÓFS á NK frumur og samspil þeirra við daufkyrninga rannsökuð, sem og myndun þeirra á lípíðafleiddum boðefnum. Kvenkyns C57Bl/6 mýs fengu viðmiðunarfæði með íbættu 2,8% af meinhaddsfiskolíu (fiskolíufæði) í samtals 5 vikur. Þær voru bólusettar tvisvar með metýleruðu nautgripaalbúmíni með tveggja vikna millibili og sama sameind síðan notuð til að mynda kviðarholsbólgu. Músum var lógað fyrir og 1,5, 3, 6 og 12 klst eftir bólgumyndun og kviðarholsvöka og -frumum safnað ásamt garnahengiseitlum. NK frumur úr mönnum voru forræktaðar með FÓFS og síðan ræktaðar áfram einar og sér eða með nýeingruðum daufkyrningum. Fjöldi daufkyrninga og NK frumna og tjáning þeirra á yfirborðssameindum var mæld með frumuflæðisjá; styrkur bólgumyndandi og bólguhjöðnunar sameinda með ELISA, Luminex og LC-MS/MS aðferðum; tjáning á lípoxýgenösum með SimpleWestern aðferð; og fjöldi frumna í stýrðum frumudauða í eitilvef með TUNEL litun. Fiskolía í fæði fækkaði daufkyrningum í kviðarholi og minnkaði tjáningu þeirra á „ekki-éta-mig“ sameindinni CD47 á sama tíma og hún fjölgaði daufkyrningum sem voru í stýrðum frumudauða miðað við það sem var í músum sem fengu viðmiðunarfæði. Fiskolíufæðið minnkaði einnig styrk bólguhvetjandi boðefna og flakkboða, s.s. CXCL1, -2, CCL20, TNF-α, IL-6, og IL-6Rα, og eikósanóíða upprunnum úr ómega-6 FÓFS, þ.e. prostaglandína og þromboxans B2. Samtímis jókst styrkur IGF-1, TGFβ1, og TNFRII í kviðarholsvökva músa sem fengu fiskolíu í fæði miðað við það sem var í músum sem fengu viðmiðunarfæði. Mýs sem fengu fiskolíufæði höfðu einnig hærra hlutfall af ómega-3 FÓFS á móti ómega-6 FÓFS í kviðarholsvökva og -frumum á öllum tímapunktum í bólgunni. Fiskolíufæði jók fjölda CD11b+CD27- NK frumna í kviðarholi músa á þeim tíma sem bólgan var í hámarki og sömuleiðis styrk CCL5, CXCL10, og CXCL12 í kviðarholsvökva. NK frumur úr músum sem fengu fiskolíufæði tjáðu meira af CCR5 en NK frumur úr músum sem fengu viðmunarfæði. Auk þess var hærra hlutfall, heildarfjöldi og tjáning á CD107a á NK frumum úr músum sem fengu fiskolíufæði en hlutfall, heildarfjöldi og tjáning á CD62L á NK frumum lægra en það sem var í NK frumum úr músum sem fengu viðmiðunarfæði. Ómega-3 fjölómettaða fitusýran dókósahexaen sýra (DHA) dró úr áhrifum NK frumna til að auka tjáningu daufkyrninga á CD11b og CD47. Þá leiddu DHA meðhöndlaðar NK frumur ekki til auknins fjölda CD11b+CD47+++ daufkyrninga eins og NK frumur án DHA meðhöndlunar gerðu. DHA meðhöndlun NK frumna dró úr getu þeirra að auka stýrðan frumudauða daufkyrninga eftir 18 klst í rækt. DHA meðhöndlun á NK frumum leiddi einnig til minni tjáningar á NKp46 í kjölfar samræktar með daufkyrningum. Að lokum leiddi DHA meðhöndlun NK frumna til minni seytunar þeirra á bólguboðefnum í kjölfar samræktar með daufkyningum. Óræstar NK frumur tjáðu 5-, 12-, and 15-lípoxýgenasa sem eru ensím sem þarf til að mynda lípíðafleidd bólguhjöðnunarboðefni. Myndun lípíðafleiddra boðefna var mismunandi eftir því hvort NK frumurnar voru meðhöndlaðar með arakídon sýru (AA), eikósapentaen sýru eða DHA. Í ljós kom að NK frumur sem voru meðhöndlaðar með ómega-3 FÓFS gátu myndað lípíðafleidd bólguhjöðnunarboðefni og forvera þeirra án hjálpar frá daufkyrningum. Að lokum kom í ljós að NK frumur sem voru meðhöndlaðar með AA gátu bara myndað prostaglandín og þromboxan ef daufkyrningar voru með í ræktinni. Niðurstöðurnar sýna að fiskolía í fæði eykur mörg af kennimerkjum bólguhjöðnunar, m.a. stýrðs frumudauða daufkyrninga, át þeirra og færslu í eitla. Þessum áhrifum getur verið miðlað af aukinni íferð frumudrápsvaldandi CD11b+CD27- NK frumna. Auk þess sýna niðurstöðurnar að DHA hefur áhrif á hvernig NK frumur eiga samskipti við daufkyrninga. Að lokum sýna þær að NK frumur tjá lípoxýgenasa og geta myndað lípíðafleidd bólguhjöðnunarboðefni sem hvetja til bólguhjöðnunar

Dietary Fish Oil Increases the Number of CD11bCD27 NK Cells at the Inflammatory Site and Enhances Key Hallmarks of Resolution of Murine Antigen-Induced Peritonitis.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadPurpose: To determine the effects of dietary omega-3 polyunsaturated fatty acids (PUFAs) on recruitment of natural killer (NK) cells and resolution responses in antigen-induced peritonitis in mice. Methods: Mice were fed fish oil-enriched or control diets, immunized twice and challenged intraperitoneally with methylated bovine serum albumin. Prior to and at different time-points following inflammation induction, expression of surface molecules on peritoneal cells was determined by flow cytometry, concentration of soluble mediators in peritoneal fluid by ELISA or Luminex, and of lipid mediators by LC-MS/MS, and number of apoptotic cells in mesenteric lymph nodes by TUNEL staining. Results: Mice fed the fish oil diet had higher number of CD11b+CD27- NK cells as well as a higher proportion of CD107a+ NK cells in their peritoneum 6 h after inflammation induction than mice fed the control diet. They also had higher numbers of CCR5+ NK cells and higher concentrations of CCL5 and CXCL12. Additionally, a higher fraction of apoptotic neutrophils but lower fraction of CD47+ neutrophils were present in the peritoneum of mice fed the fish oil diet 6 h after inflammation induction and the fish oil fed mice had a shorter resolution interval. They also had lower concentrations of pro-inflammatory mediators but higher concentrations of the anti-inflammatory/pro-resolution mediators TGF-β, IGF-1, and soluble TNF RII, as well as higher ratios of hydroxyeicosapentaenoic acid (HEPE) to hydroxyeicosatetraenoic acid (HETE) than mice fed the control diet. Conclusion: The results demonstrate that dietary fish oil increases the number of mature NK cells at the inflamed site in antigen-induced peritonitis and enhances several key hallmarks of resolution of inflammation, casting light on the potential mechanisms involved. Keywords: apoptosis; lipid mediators; natural killer cells; neutrophils.Icelandic Research Fund University of Iceland Research Fund Landspitali University Hospital Research Fund Memorial Fund of Helga Jonsdottir and Sigurlidi Kristjansso