TNF Production Research Articles

Aloe Metabolites Prevent LPS-Induced Sepsis and Inflammatory Response by Inhibiting Mitogen-Activated Protein Kinase Activation.

Aloe, a polyphenolic anthranoid-containing Aloe vera leaves, is a Chinese medicine and a popular dietary supplement worldwide. In in vivo situations, polyphenolic anthranoids are extensively broken down into glucuronides and sulfate metabolites by the gut and the liver. The anti-inflammatory potential of aloe metabolites has not been examined. The aim of this study was to investigate the anti-inflammatory effects of aloe metabolites from in vitro (lipopolysaccharides (LPS)-activated RAW264.7 macrophages) and ex vivo (LPS-activated peritoneal macrophages) to in vivo (LPS-induced septic mice). The production of proinflammatory cytokines (TNF-[Formula: see text] and IL-12) and NO was determined by ELISA and Griess reagents, respectively. The expression levels of iNOS and MAPKs were analyzed by Western blot. Our results showed that aloe metabolites inhibited the expression of iNOS, decreased the production of TNF-[Formula: see text], IL-12, and NO, and suppressed the phosphorylation of MAPKs by LPS-activated RAW264.7 macrophages. In addition, aloe metabolites reduced the production of NO, TNF-[Formula: see text] and IL-12 by murine peritoneal macrophages. Furthermore, aloe administration significantly reduced the NO level and exhibited protective effects against sepsis-related death in LPS-induced septic mice. These results suggest that aloe metabolites exerted anti-inflammatory effects in vivo, and that these effects were associated with the inhibition of inflammatory mediators. Therefore, aloe could be considered an effective therapeutic agent for the treatment of sepsis.

An Oligodeoxynucleotide with AAAG Repeats Significantly Attenuates Burn-induced Systemic inflammatory Responses by inhibiting interferon Regulatory Factor 5 Pathway

Previously, we showed that an oligodeoxynucleotide (ODN) with AAAG repeats (AAAG ODN) rescued mice from fatal acute lung injury (ALI) induced by influenza virus and inhibited production of tumor necrosis factor-α (TNF-α) in the injured lungs. However, its underlying mechanisms remain to be elucidated. Upon the bioinformatic analysis revealing that the sequence of AAAG ODN is in consensus with the interferon regulatory factor 5 (IRF5) binding site in the cis-regulatory elements of proinflammatory cytokines, we tried to explore whether AAAG ODN could attenuate burn injury-induced systemic inflammatory responses by inhibiting the IRF5 pathway. Using a mouse model with sterile systemic inflammation induced by burn injury, we found that AAAG ODN prolonged the lifespan of the mice, decreased the expression of IRF5 in the injured skin, reduced the production of TNF-α and IL-6 in the blood and injured skin, and attenuated the ALI. These effects were correlated with AAAG ODN-mediated inhibition of nuclear translocation of IRF5. The data suggest that AAAG ODN could act as a cytoplasmic decoy capable of interfering the function of IRF5 and be developed as a drug candidate for the treatment of inflammatory diseases.

포도가지와 새송이버섯 혼합 추출물의 항염증과 아토피 피부염 개선 상승효과

본 연구는 캠벨어얼리 포도가지와 새송이버섯 혼합 추출물이 활성화된 면역세포에서 항염증 및 화학항원과 생물학적 항원을 동시에 처리한 아토피 피부염 유사 모델 마우스에서 아토피 개선에 대한 상승효과를 알아보았다. 그 결과 지방질다당류로 자극된 RAW 264.7 세포에 포도가지와 새송이버섯 각각의 추출물 처리했을 때 보다 이들 추출물을 혼합하여 처리할 때 산화질소와 <TEX>$PGE_2$</TEX> 생성을 효과적으로 억제하였다. 또한 지방질다당류로 자극된 RAW 264.7 세포와 PMA와 A23187로 활성화된 HMC-1 세포에서도 각각의 추출물 보다 혼합추출물을 처리했을 때 <TEX>$TNF-{\alpha}$</TEX>와 IL-6 생성에 대한 억제 효과가 매우 우수하였다. 더욱이 DNCB와 집먼지진드기 항원으로 유도된 아토피 피부염 유사 모델에서 포도가지와 새송이버섯 혼합 추출물 투여는 각각의 추출물 투여에 비해서 외부적 피부병변과 피부두께 및 피부조직학적 등 임상적으로 아토피 피부염에 대한 개선 효과가 매우 뛰어남을 확인할 수 있었다. 이러한 포도가지와 새송이버섯 혼합 추출물의 아토피 피부염 개선 효과는 혈청 내 IgE와 IL-4의 생성을 억제함으로써 각각의 추출물에 비해서 상승되는 효과가 있음을 나타내 주었다. 이러한 결과는 포도가지와 새송이버섯 각각의 추출물 투여보다는 이들의 추출물을 혼합하여 투여하는 것이 아토피 피부염을 개선시키는데 효과적임을 알 수 있었다. The aim of this study was to investigate the synergistic anti-inflammatory and anti-dermatitis effects of grape branch extract (GBE) and Pleurotus eryngii (PEE) combinations on the active immune cells and atopic dermatitis-like skin lesions in mice. The results showed that the combination of GBE (<TEX>$12.5{\mu}g/mL$</TEX>) and PEE (<TEX>$500{\mu}g/mL$</TEX>) led to much stronger inhibitory effects on the production of inflammatory mediators, such as NO and <TEX>$PGE_2$</TEX> than that exhibited by GBE (<TEX>$25{\mu}g/mL$</TEX>) and PEE (<TEX>$1000{\mu}g/mL$</TEX>) alone, even at higher concentrations, in LPS-stimulated RAW 264.7 macrophages. The combination of GBE and PEE synergistically inhibited the production of TNF- and IL-6 in LPS-stimulated RAW 264.7 macrophages and PMA plus A23187-activated HMC-1 cells. Furthermore, combined GBE and PEE had a stronger ameliorative effect than GBE and PEE alone by inhibiting the clinical sores, IgE, and IL-4 on atopic dermatitis-like skin lesions in mice. Collectively, these results suggested that the combination of GBE and PEE produced a synergistic anti-inflammatory and anti-atopic dermatitis effect on immune cells and atopic dermatitis-like skin lesions in mice.

Epoxyeicosanoids prevent intervertebral disc degeneration in vitro and in vivo.

Intervertebral disc (IVD) degeneration is considered a common cause of low back pain. In the degenerating IVD, the production of pro-inflammatory cytokines, including IL-1 and TNF-α, progressively increases, contributing to the degenerative process. Epoxyeicosatrienoic acids (EETs), synthesized from arachidonic acid by cytochrome P450 enzymes, act as autocrine and paracrine effectors in regulating inflammation, cardiovascular functions, and angiogenesis. EETs were shown to be especially potent promoters of tissue regeneration. Considering their anti-inflammatory and anti-catabolic potential, we investigated whether EETs can influence IVD degeneration. We found that 14,15-EET protected rat nucleus pulposus (NP) cells against death induced by treatment with H2O2and TNF-α in vitro. At the molecular level, 14,15-EET significantly inhibited the NF-κB pathway, which plays essential roles in the degeneration and survival of NP cells. As a result, 14,15-EET efficiently prevented the matrix remodeling response of NP cells to TNF-α. Using a needle-punctured rat tail model, the influence of 14,15-EET on IVD degeneration in vivo was evaluated using radiographs, magnetic resonance images (MRI), and histological analysis. We observed that 14,15-EET prevented IVD degeneration. Our findings demonstrated that 14,15-EET can enhance the survival of NP cells and inhibit IVD degeneration. The EET pathway may be a novel therapeutic target against IVD degeneration.

Inflammation and exercise: Inhibition of monocytic intracellular TNF production by acute exercise via β2-adrenergic activation

Regular exercise is shown to exert anti-inflammatory effects, yet the effects of acute exercise on cellular inflammatory responses and its mechanisms remain unclear. We tested the hypothesis that sympathoadrenergic activation during a single bout of exercise has a suppressive effect on monocytic cytokine production mediated by β2 adrenergic receptors (AR). We investigated the effects of 20-min moderate (65–70% VO2 peak) exercise-induced catecholamine production on LPS-stimulated TNF production by monocytes in 47 healthy volunteers and determined AR subtypes involved. We also examined the effects of β-agonist isoproterenol and endogenous β- and α-agonists epinephrine and norepinephrine, and receptor-subtype-specific β- and α-antagonists on TNF production in a series of in vitro investigations. LPS-stimulated TNF production by peripheral blood monocytes was determined intracellularly by flow cytometry, using an intracellular protein transport inhibitor. Percent TNF-producing monocytes and per-cell TNF production with and without LPS was suppressed by exercise with moderate to large effects, which was reversed by a β2-AR antagonist in spite that plasma TNF levels did not change. This inhibitory response in TNF production by exercise was mirrored by β-AR agonists in an agonist-specific and dose-dependent manner in vitro: similar isoproterenol (EC50=2.1–4.7×10−10M) and epinephrine (EC50=4.4–10×10−10M) potency and higher norepinephrine concentrations (EC50=2.6–4.3×10−8M) needed for the effects. Importantly, epinephrine levels observed during acute exercise in vivo significantly inhibited TNF production in vitro. The inhibitory effect of the AR agonists was abolished by β2-, but not by β1- or α-AR blockers. We conclude that the downregulation of monocytic TNF production during acute exercise is mediated by elevated epinephrine levels through β2-ARs. Decreased inflammatory responses during acute exercise may protect against chronic conditions with low-grade inflammation.

CD141 Expressing Monocytes Show an Inflammatory Profile and Are Associated with Low-Risk Features in Myelodysplastic Syndromes

IntroductionImmune responses play an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). It has been shown that a consistent immunological signature in MDS comprises an increase in Th17 cells in low risk MDS and an expansion of T regulatory cells (Tregs) in high risk subtypes. Additionally, the presence of high Treg numbers in lower risk stages is an independent prognostic value and is associated with poor survival. The high pro-inflammatory response in low risk MDS is also linked to autoimmune features in these patients. Identification of immune cells that could potentially contribute to the inflammatory environment are of interest to prevent disease progression and, on the other hand, restore excessive immune responses by therapeutic interaction. The exact role of monocytes in this early immune disturbance is yet to be understood. Our preliminary findings showed that MDS-derived monocytes have elevated expression levels of the dendritic cell marker BDCA3/CD141 compared to healthy monocytes. The aim of this study was therefore to investigate the phenotypic and functional characteristics of CD141+ versus CD141- monocytes in MDS patients and compare them with healthy monocytes.Patients and methodsMonocytes were gated as CD14hiCD11chiHLA-DR+. The percentage of CD141+ monocytes was determined in 143 MDS bone marrow (BM) and 22 normal bone marrow (NBM) samples, and in the peripheral blood (PB) of 33 MDS patients and 28 normal PB (NPB) samples. Furthermore, CD141 expression was assessed in different disease stages and correlated to several disease characteristics such as the presence of ring sideroblasts (RS), blast percentage and cytogenetic risk group. Additionally, sorted CD141- and CD141+ monocytes of two MDS patients were tested for their functional capacities, including their ability to skew T cells and to up-regulate the co-stimulatory molecules CD80 and CD86 upon stimulation. The production of different cytokines was measured in culture supernatants by using Cytometric Bead Array (CBA).ResultsThe percentage of CD141+ monocytes was highly increased in MDS BM compared to NBM (36% vs 12%, p<0.0001), and, also in MDS PB compared to NPB (24% vs 11%, p=0.012). Additionally, the presence of CD141+ monocytes in PB and BM was positively correlated between the two compartments (r= 0.82, p<0.0001). The increased percentage of BM-derived CD141+ monocytes was more evident in low risk groups (RA, RARS and RCMD) than in high risk groups ([RAEB-I/II], 39% vs 22%, p<0.001), and hence, was associated with low blast counts (<5% vs ≥5% blasts: 40% vs 22%, p=0.004). The presence of RS was also correlated with elevated rates of CD141+ monocytes (50% vs 28%, p=0.002). In addition, MDS patients that showed a very good-to-good cytogenetic risk profile had increased frequencies of CD141+ monocytes compared to patients in poor and very poor risk groups (36% vs 22%, p=0.013). Phenotypic analyses showed elevated expression levels of HLA-DR on CD141+ monocytes compared to total monocytes (1.5 fold, p<0.001). Preliminary functional tests revealed no differences in up-regulation of CD80 and CD86 upon stimulation in CD141+ or CD141- MDS monocytes. However, there was a trend towards higher production of IL-1β, IL-6 and TNF and a reduction in FoxP3+ Treg skewing in cultures of CD141+ monocytes compared to CD141- monocytes.ConclusionIn conclusion, CD141+ monocytes were found in much higher frequencies in the BM and PB of MDS patients compared to healthy control BM and PB. Their presence was associated with a low risk stage of the disease, low blast counts, the presence of RS and a very good-to-good cytogenetic risk profile. The first functional analyses point to a high pro-inflammatory state of CD141+ compared to CD141- monocytes. Therefore we hypothesize that these monocytes may contribute to an immune stimulatory environment in low risk MDS patients with potentially a favourable prognostic value. DisclosuresNo relevant conflicts of interest to declare.

Placental Growth Factor (PlGF) Enhances TLR/MK2 Dependent TNF Gene Transcription and Dissociates miRNAs Targeting TNF Regulatory Transcripts from the Polysome

Introduction:Increasing evidence has demonstrated a cooperative link between angiogenic stimuli and inflammatory responses, though molecular and cellular mechanisms involved in this effect are unclear. Pathologic increases in one such angiogenic factor, placental growth factor (PlGF), a member of the vascular endothelial growth factor family, have been described in diverse clinical settings characterized by an increased risk of inflammatory complications (hemoglobinopathies, inflammatory arthritis, inflammatory bowel disease, and allogeneic hematopoietic cell transplantation). We recently confirmed our hypothesis that the effect of PlGF on the inflammatory response is the result of its capacity to enhance toll-like receptor (TLR)-dependent production of inflammatory cytokines in mononuclear phagocytes (Blood 2015 126:1006). Seeking to identify signaling pathways involved in this cooperative effect, we used TNF gene expression as the paradigm. Of the signaling pathways tested, the most profound effect of PlGF was on the state of activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2). We used loss-of-function approaches, polysome profiling, and microarray to test the hypothesis that PlGF amplifies TLR-dependent TNF gene expression in an MK2-dependent manner and to determine whether the MK2 effect was the result of its known capacity to influence the stability and translation of TNF mRNA.Methods:Primary normal donor peripheral blood CD14+ cells were exposed to PlGF (250 ng/ml) and the TLR-7/8 ligand R848 (3 μM), in the presence or absence of small molecule inhibitors of MK2. Secreted TNF protein was measured at 24-hours by ELISA, and mRNA expression at 4-6 hours determined by microarray and qRT-PCR. Actinomycin D chase experiments were performed to determine the effect of PlGF on TNF mRNA decay. RNA from polysome fractions of primary CD14+cells were separated using sucrose density gradient sedimentations, and tested using Affymetrix expression microarrays.Results:PlGF alone had no effect on TNF secretion by primary CD14+ cells. However, CD14+ cells exposed to both PlGF and R848 produced significantly more TNF protein than cells exposed to R848 alone (mean 2.35-fold, p=0.0002, n=26). PlGF significantly enhanced R848-induced TNF mRNA expression, compared to R848 alone (mean 2.46-fold, p=0.03, n=12). Immunoblotting demonstrated that: (a) PlGF alone enhanced MK2 phosphorylation compared to untreated cells, and (b) PlGF markedly enhanced R848-induced phosphorylation of MK2 (Figure). While PlGF enhanced total TNF mRNA expression in primary CD14+cells treated with R848, there was no effect of PlGF on TNF mRNA stability in Actinomycin D chase experiments, and no increase in polysome bound TNF mRNA. However, PlGF exposure reduced multiple miRNAs in the polysome fraction including those predicted to target transcription factors known to enhance TNF expression. Microarray confirmed that PlGF increased the abundance of most of those factors, specifically EBF1, EGR1, FOS, NFKB1, and REL. Treatment of primary cells with an MK2 inhibitor (PF-3644022, 1 μM) blocked the effect of PlGF on R848-induced TNF mRNA and protein production.Conclusions:PlGF significantly increases TNF production by primary CD14+ cells, and it does so, at least in part, by activating the p38 MAPK/MK2 signaling pathway. Notwithstanding the well-known capacity of MK2 to influence gene expression post-transcriptionally, the mechanism of the PlGF/TLR effect on TNF expression is unambiguously at the point of transcription and independent of TNF mRNA stability or translation, suggesting a novel role for PlGF-dependent MK2 activation in modulating TNF gene transcription. Based on our findings, we hypothesize that PlGF-dependent MK2 activation enhances TNF transcription indirectly by enhancing stability and translation of well-known TNF transcription factors, and this effect involves the PlGF/MK2 dependent disruption of mRNA/miRNA complexes in polysomes, resulting in enhanced stability and translation of the transcription factors EBF1, EGR1, FOS, NFKB1, and REL. These results reveal a previously unrecognized mechanism by which PlGF primes innate immune inflammatory responses to TLR-pathway activation. MK2 inhibitors should be evaluated in preclinical models as a potential treatment option for inflammatory complications in patients with high PlGF states. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DETC-based bacterial cellulose bio-curatives for topical treatment of cutaneous leishmaniasis.

The treatment of leishmaniasis still relies on drugs with potentially serious adverse effects. Herein, we tested a topical formulation of bacterial cellulose (BC) membranes containing Diethyldithiocarbamate (DETC), a superoxide dismutase 1 inhibitor. Leishmania-infected macrophages exposed to BC-DETC resulted in parasite killing, without pronounced toxic effects to host cells. This outcome was associated with lower SOD1 activity and higher production of superoxide and cytokine mediators. Topical application of BC-DETC significantly decreased lesion size, parasite load and the inflammatory response at the infection site, as well as the production of both IFN-γ and TNF. Combination of topical BC-DETC plus intraperitoneal Sbv also significantly reduced disease development and parasite load. The leishmanicidal effect of BC-DETC was extended to human macrophages infected with L. braziliensis, highlighting the feasibility of BC-DETC as a topical formulation for chemotherapy of cutaneous leishmaniasis caused by L. braziliensis.

Reduced expression of monocyte CD200R is associated with enhanced proinflammatory cytokine production in sarcoidosis.

In sarcoidosis, the proinflammatory cytokines interferon gamma, tumour necrosis factor and interleukin-6 are released by monocyte-derived macrophages and lymphocytes in the lungs and other affected tissues. Regulatory receptors expressed on monocytes and macrophages act to suppress cytokine production, and reduced expression of regulatory receptors may thus promote tissue inflammation. The aim of this study was to characterise the role of regulatory receptors on blood monocytes in patients with sarcoidosis. Cytokine release in response to stimulation of whole blood was measured in healthy controls and Caucasian non-smoking patients with sarcoidosis who were not taking disease modifying therapy. Expression of the regulatory molecules IL-10R, SIRP-α/β, CD47, CD200R, and CD200L was measured by flow cytometry, and functional activity was assessed using blocking antibodies. Stimulated whole blood and monocytes from patients with sarcoidosis produced more TNF and IL-6 compared with healthy controls. 52.9% of sarcoidosis patients had monocytes characterised by low expression of CD200R, compared with 11.7% of controls (p < 0.0001). Patients with low monocyte CD200R expression produced higher levels of proinflammatory cytokines. In functional studies, blocking the CD200 axis increased production of TNF and IL-6. Reduced expression of CD200R on monocytes may be a mechanism contributing to monocyte and macrophage hyper-activation in sarcoidosis.

Age-Specific Adjuvant Synergy: Dual TLR7/8 and Mincle Activation of Human Newborn Dendritic Cells Enables Th1 Polarization.

Due to functionally distinct cell-mediated immunity, newborns and infants are highly susceptible to infection with intracellular pathogens. Indeed, neonatal Ag-presenting dendritic cells (DCs) demonstrate impaired Th1 responses to many candidate adjuvants, including most TLR agonists (TLRAs). Combination adjuvantation systems may provide enhanced immune activation but have typically been developed without regard to the age of the target population. We posited that distinct combinations of TLRAs and C-type lectin receptor agonists may enhance Th1 responses of newborn DCs. TLRA/C-type lectin receptor agonist combinations were screened for enhancement of TNF production by human newborn and adult monocyte-derived DCs cultured in 10% autologous plasma or in newborn cord, infant, adult, and elderly whole blood. Monocyte-derived DC activation was characterized by targeted gene expression analysis, caspase-1 and NF-κB studies, cytokine multiplex and naive autologous CD4+ T cell activation. Dual activation of newborn DCs via the C-type lectin receptor, macrophage-inducible C-type lectin (trehalose-6,6-dibehenate), and TLR7/8 (R848) greatly enhanced caspase-1 and NF-κB activation, Th1 polarizing cytokine production and autologous Th1 polarization. Combined activation via TLR4 (glycopyranosyl lipid adjuvant aqueous formulation) and Dectin-1 (β-glucan peptide) acted synergistically in newborns and adults, but to a lesser extent. The degree of synergy varied dramatically with age, and was the greatest in newborns and infants with less synergy in adults and elders. Overall, combination adjuvant systems demonstrate markedly different immune activation with age, with combined DC activation via Macrophage-inducible C-type lectin and TLR7/8 representing a novel approach to enhance the efficacy of early-life vaccines.

Cell-Extrinsic TNF Collaborates with TRIF Signaling To Promote Yersinia-Induced Apoptosis.

Innate immune responses that are crucial for control of infection are often targeted by microbial pathogens. Blockade of NF-κB and MAPK signaling by the Yersinia virulence factor YopJ inhibits cytokine production by innate immune cells but also triggers cell death. This cell death requires RIPK1 kinase activity and caspase-8, which are engaged by TLR4 and the adaptor protein TRIF. Nevertheless, TLR4- and TRIF-deficient cells undergo significant apoptosis, implicating TLR4/TRIF-independent pathways in the death of Yersinia-infected cells. In this article, we report a key role for TNF/TNFR1 in Yersinia-induced cell death of murine macrophages, which occurs despite the blockade of NF-κB and MAPK signaling imposed by Yersinia on infected cells. Intriguingly, direct analysis of YopJ injection revealed a heterogeneous population of injection-high and injection-low cells, and demonstrated that TNF expression came from the injection-low population. Moreover, TNF production by this subpopulation was necessary for maximal apoptosis in the population of highly injected cells, and TNFR-deficient mice displayed enhanced susceptibility to Yersinia infection. These data demonstrate an important role for collaboration between TNF and pattern recognition receptor signals in promoting maximal apoptosis during bacterial infection, and demonstrate that heterogeneity in virulence factor injection and cellular responses play an important role in promoting anti-Yersinia immune defense.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3−/− and ASC−/−mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1β, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated β-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Activation and increase of radio-sensitive CD11b+ recruited Kupffer cells/macrophages in diet-induced steatohepatitis in FGF5 deficient mice.

We have recently reported that Kupffer cells consist of two subsets, radio-resistant resident CD68+ Kupffer cells and radio-sensitive recruited CD11b+ Kupffer cells/macrophages (Mφs). Non-alcoholic steatohepatitis (NASH) is characterized not only by hepatic steatosis but also chronic inflammation and fibrosis. In the present study, we investigated the immunological mechanism of diet-induced steatohepatitis in fibroblast growth factor 5 (FGF5) deficient mice. After consumption of a high fat diet (HFD) for 8 weeks, FGF5 null mice developed severe steatohepatitis and fibrosis resembling human NASH. F4/80+ Mφs which were both CD11b and CD68 positive accumulated in the liver. The production of TNF and FasL indicated that they are the pivotal effectors in this hepatitis. The weak phagocytic activity and lack of CRIg mRNA suggested that they were recruited Mφs. Intermittent exposure to 1 Gy irradiation markedly decreased these Mφs and dramatically inhibited liver inflammation without attenuating steatosis. However, depletion of the resident subset by clodronate liposome (c-lipo) treatment increased the Mφs and tended to exacerbate disease progression. Recruited CD11b+ CD68+ Kupffer cells/Mφs may play an essential role in steatohepatitis and fibrosis in FGF5 null mice fed with a HFD. Recruitment and activation of bone marrow derived Mφs is the key factor to develop steatohepatitis from simple steatosis.

During acute experimental infection with the reticulotropic Trypanosoma cruzi strain Tulahuen IL-22 is induced IL-23-dependently but is dispensable for protection.

Protective immunity against Trypanosoma cruzi, the causative agent of Chagas disease, depends on the activation of macrophages by IFN-γ and IL-17A. In contrast, IL-10 prevents immunopathology. IL-22 belongs to the IL-10 cytokine family and has pleiotropic effects during host defense and immunopathology, however its role in protection and pathology during T. cruzi infection has not been analyzed yet. Therefore, we examined the role of IL-22 in experimental Chagas disease using the reticulotropic Tulahuen strain of T. cruzi. During infection, IL-22 is secreted by CD4-positive cells in an IL-23-dependent fashion. Infected IL-22−/− mice exhibited an increased production of IFN-γ and TNF and displayed enhanced numbers of activated IFN-γ-producing T cells in their spleens. Additionally, the production of IL-10 was increased in IL-22−/− mice upon infection. Macrophage activation and by association the parasitemia was not affected in the absence of IL-22. Apart from a transient increase in the body weight loss, infected IL-22−/− mice did not show any signs for an altered immunopathology during the first fourteen days of infection. Taken together, although IL-22 is expressed, it seems to play a minor role in protection and pathology during the acute systemic infection with the reticulotropic Tulahuen strain of T. cruzi.

Abstract # 1863 What your monocytes’ inflammatory responses to adrenergic stimulation can tell you about your risk for cardiovascular disease beyond blood pressure

Pathophysiology of hypertension includes hyperactive sympathoadrenal system and elevated inflammation, but the link between the two and its clinical risk remain unclear. Subclinically elevated blood pressure (BP), pre-hypertension (PHT), is related to future cardiovascular disease (CVD) and greater mortality. In 177 adults with PHT (125 n = 66) versus normal BP (NBP, n = 105), we investigated the association between monocytes’ sensitivity to isoproterenol control of LPS-induced TNF production using flow cytometry and CV risk calculated using Framingham risk score (FRS) for CVD. FRSs, either factoring in BMI or lipid levels, were greater in PHT than NBP group ( p ′ s r = −.22 to −.25, p ′ s r = −.10 to −.20). Our results indicate that even mildly elevated BP is related to impaired inflammation regulation mediated by sympathoadrenal over-activation. Meanwhile, the characteristics of the associations of BP vs. inflammation regulation to CVD risk appear different.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low-Affinity Memory CD8+ T Cells during Heterologous Immunity.

The affinity of a TCR binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low-affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic Ag and mediate graft rejection. We found that low-affinity-primed memory CD8(+) T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared with high-affinity-primed memory T cells. Low-affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2(hi) proliferating cells. Low-affinity-primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low- but not high-affinity-primed animals. These data establish a functional connection between TNF signaling and TCR-priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation.

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates—a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably contributing to the immunopathogenesis of the disease.

Mucosal-associated invariant T cells are reduced and functionally immature in the peripheral blood of primary Sjögren's syndrome patients.

The frequencies, immunophenotype, and function of mucosal-associated invariant T (MAIT) cells were studied in patients with primary Sjögren syndrome (pSS) and healthy controls. MAIT cells were significantly decreased in the peripheral blood (PB) of patients with pSS. Vα7.2+ MAIT cells were detected in the salivary gland tissue from pSS patients, but not in controls, indicating that the reduction of MAIT cells in PB might be due to migration into the target tissue. Furthermore, the residual peripheral blood MAIT cells in pSS patients showed altered immunophenotype and function. While MAIT cells from controls were almost exclusively CD8+ and expressed an effector memory immunophenotype, in pSS patients they were enriched in CD4+ and naïve subpopulations. Consistently, the functional studies demonstrated that MAIT cells from pSS showed a lower level of activation with reduced expression of CD69 and CD154 (CD40L), and a lower production of TNF and IFN-γ. In summary, our findings demonstrate that MAIT cells were reduced and phenotypically and functionally altered in PB of pSS patients. The altered function of MAIT cells in target tissues from pSS patients may result in dysregulation of mucosal immunity leading to microbial damage of mucosal surfaces and subsequent initiation of autoimmune response.

Anti-TNF Therapy.

Tumor necrosis factor (TNF) is one of the most important cytokines produced by macrophages. TNF is a very important component of host defense, released very rapidly after all types of injuries and stimuli. The kinetics of TNF release are short, and so it is perhaps not surprising that prolonged TNF production is associated with pathology. This was first elucidated in rheumatoid arthritis but extends to other chronic inflammatory diseases such as Crohn's disease and psoriasis. In this chapter, the discovery of anti-TNF therapy is reviewed, with its benefit but also its limitations. The potential of anti-TNF therapy in other diseases, e.g., cardiovascular and fibrosis, is discussed, as is the opportunity to define ways of blocking TNF synthesis.

THE EFFECT OF S-ADENOSYLMETHIONINE ON AMELIORATING Aβ INDUCED NEURAL INJURY

β-amyloid peptide (Aβ), as a kind of strong oxidative stress inducing agent, is the main cause of Alzheimer's disease (AD). S-adenosylmethionine (SAM) is a bioactive substance existing widely in the body. SAM itself can be used as a methyl donor to regulate gene expression through epigenetic modification. Meanwhile glutathione produced in SAM metabolic process can increase the capacity of antioxidant. It is reported that significant low SAM is found in the cerebrospinal fluid in AD patients. The current study aimed at investigating the neuroprotective role of SAM on Aβ induced neural injury. Aβ intrahippocampal injection rat model and human neuroblastoma SH-SY5Y cells treated with Aβ were used to explore the effect of SAM on cellular viability, apoptosis, the changes of ROS, NO/iNOS, MDA, GSH, and SOD. The expression profiles of all BDNF transcripts were examined by qPCR and Western blot. Also the spatial capacity was tested by Morris maze. SAM could improve the cellular viability by inhibition of apoptosis. SAM could reverse the oxidative stress damage induced by Aβ through reduction of ROS and NO/iNOS production, and enhancement of GSH and SOD. Moreover SAM could reduce the production of IL-6 and TNF by inhibition of the activation of microglia and astrocyte induced by Aβ in vivo. SAM could improve the low BDNF expression under Aβ insult mainly through BDNF exon IV and exon VI. SAM could improve spatial capacity of rats under Aβ insult. SAM might play a neuroprotective role by increasing the expression of endogenous BDNF gene, inhibition of oxidative stress and inflammatory reaction. SAM may be a potential target for therapy to AD.