Aberrant up-regulation of iNOS/NO system is correlated with an increased abundance of Foxp3+ cells and reduced effector/memory cell markers expression during colorectal cancer: immunomodulatory effects of cetuximab combined with chemotherapy.

Colorectal cancer (CRC) remains the most cancer type related to chronic inflammation; however, the mechanisms that link inflammation to CRC development and progression are still poorly understood. Our study aimed to investigate one of the prominent inflammatory response in cancers, iNOS/NO system. In this regard, we evaluated the link between the iNOS/NO system and CRC progression, its relation with the host immune responses and its response to cetuximab combined with chemotherapy. We found that the nitrite levels were nearly twice as high in metastatic CRC plasma and culture supernatants from PBMCs and tumor explants compared with those without metastases and healthy controls. Interestingly, we showed that the highest iNOS expression and NO levels are present in the damaged CRC tissues that have highest leukocyte infiltration. Our findings highlight the implication of iNOS/NO system in tissue alteration and leukocyte invasion. Thus, we observed imbalance between effector/memory T cell markers and Treg transcription factor (Foxp3). Accordingly, we detected higher IFNγ and T-bet expression levels in colorectal tumor tissues at early stage. In contrast, consistent with iNOS and Foxp3 expression, TGFß, CTLA-4 and IL-10 were significantly related to the tumor stage progression. Furthermore, our study revealed that Cetuximab combined with chemotherapy treatment markedly down-regulates iNOS/NO system as well as IL-10 and TGFß levels. Altogether, we conclude that cetuximab can potentiate the efficacy of chemotherapy, particularly by iNOS/NO system and immunosuppressive cytokines modulation. Thus, we suggest that iNOS/NO system may represent an attractive candidate biomarker for monitoring CRC progression, malignity and response to therapy.

Interferon-ß inhibits inflammatory responses mediators via suppression of iNOS signaling pathway in PBMCs from patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) represents a chronic, systemic autoimmune disorder, characterized by lymphocytic infiltration of exocrine glands, inducing compromised secretory function and tissue destruction. Increasing evidence had revealed that inflammatory mediators, such as nitric oxide (NO) and pro-inflammatory cytokines, are critical in the development and perpetuation of pSS systemic manifestations. In our current study, we aimed to investigate the ex vivo immunomodulatory effect of interferon (IFN)-ß on iNOS expression, as well as on pro-inflammatory (tumor necrosis factor (TNF)-α, interleukin (IL)-6) and immunoregulatory (IL-10) cytokine production. Furthermore, we examined potential associations between the influence of IFN-ß treatment on NO production, and pSS clinical and serological manifestations. METHODS: In 41 pSS patients documented for their clinical and serological features, NO and cytokines levels were measured by the Griess method and enzyme-linked immunosorbent assay, respectively. Inducible nitric oxide synthase expression was analyzed by fluorescence immunostaining assay, using peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and pSS patients. RESULTS: Our results revealed a strong down-modulating effect of IFN-ß in the secretion of pro-inflammatory mediators including TNF-α, IL-6, and NO production. Interestingly, IFN-ß exerts an increase in IL-10 levels. The most suppressive effect exerted by IFN-ß on NO production was importantly reported for patients with neurological manifestation. This immunomodulatory effect of IFN-ß on NO production is highly related to the decrease of inducible nitric oxide synthase (iNOS) expression. CONCLUSION: Our findings highlight a consistent ex vivo inhibitory effect of IFN-ß on pro-inflammatory cytokine production and NO pathway in pSS patients. Our data suggest that IFN-ß could represent a potential candidate for targeting inflammation during pSS.

Ex vivo immunomodulatory effect of ethanolic extract of propolis during Celiac Disease: involvement of nitric oxide pathway.

Celiac Disease (CeD) is a chronic immune-mediated enteropathy, in which dietary gluten induces an inflammatory reaction, predominantly in the duodenum. Propolis is a resinous hive product, collected by honeybees from various plant sources. Propolis is well-known for its anti-inflammatory, anti-oxidant and immunomodulatory effects, due to its major compounds, polyphenols and flavonoids. The aim of our study was to assess the ex vivo effect of ethanolic extract of propolis (EEP) upon the activity and expression of iNOS, along with IFN-γ and IL-10 production in Algerian Celiac patients. In this context, PBMCs isolated from peripheral blood of Celiac patients and healthy controls were cultured with different concentrations of EEP. NO production was measured using the Griess method, whereas quantitation of IFN-γ and IL-10 levels was performed by ELISA. Inducible nitric oxide synthase (iNOS) expression, NFκB and pSTAT-3 activity were analyzed by immunofluorescence assay. Our results showed that PBMCs from Celiac patients produced high levels of NO and IFN-γ compared with healthy controls (HC). Interestingly, EEP reduced significantly, NO and IFN-γ levels and significantly increased IL-10 levels at a concentration of 50 µg/mL. Importantly, EEP downmodulated the iNOS expression as well as the activity of NFκB and pSTAT-3 transcription factors. Altogether, our results highlight the immunomodulatory effect of propolis on NO pathway and on pro-inflammatory cytokines. Therefore, we suggest that propolis may constitute a potential candidate to modulate inflammation during Celiac Disease and has a potential therapeutic value.

All-Trans Retinoic Acid Modulates TLR4/NF-κB Signaling Pathway Targeting TNF-α and Nitric Oxide Synthase 2 Expression in Colonic Mucosa during Ulcerative Colitis and Colitis Associated Cancer.

Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF-κB signaling in development and progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-α induction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.

All-trans-retinoic acid modulates nitric oxide and interleukin-17A production by peripheral blood mononuclear cells from patients with Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD), the most common form of dementia in the elderly, is a neurodegenerative disorder associated with a complex pathophysiology. It is accepted that inflammation contributes to the pathogenesis of AD. All-trans-retinoic acid (ATRA) is a bioactive derivative of vitamin A that has shown immunomodulatory effects in many immune disorders. OBJECTIVES: In our study, we aimed to investigate in vitro immunomodulatory effects of ATRA on inducible nitric oxide synthase (iNOS) expression and interleukin-17A production during AD. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from 30 Algerian AD patients and 14 age-matched nondemented controls were treated (or not) with ATRA. Production of NO and IL-17A in culture media was measured by the modified Griess method and enzyme-linked immunosorbent assay, respectively. Expression of iNOS in PBMCs was examined by fluorescence immunostaining. RESULTS: Our results showed higher spontaneous in vitro production of NO related to overexpression of iNOS in AD patients compared to controls. Remarkably, ATRA treatment showed an important downregulatory effect on NO production and iNOS expression in patients. This effect was associated with a reduction in IL-17A production and increased IL-10 release. CONCLUSIONS: Taken together, our results indicate that ATRA exerts anti-inflammatory effects in AD. Furthermore, ATRA represents a promising tool for monitoring inflammatory responses associated with disease progression.

Neuroinflammatory Responses Occur in Brain Lesions During Alzheimer's Disease: Postmortem Case Report.

BACKGROUND: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. It is characterized by a gradual decrease in cognitive function and is considered a disorder in which the intensifying neuronal loss. The autopsy is considered the gold standard for the diagnosis of AD and non-AD dementia. OBJECTIVE: Our study aims to clarify the involvement of neuroinflammation processes in brain lesions of AD. METHODS: The defunct was admitted to the forensic medicine department of Issad Hassani Hospital (Algeria). In order to recover the brain, an autopsy was performed within 24 hours of death and then immediately fixed in formaldehyde to maintain structural brain integrity for histological and immunohistochemical analysis. RESULTS: Our findings indicate the presence of tissue lesions in the specific brain regions: right middle frontal gyrus, right cingulate gyrus, right putamen and globus pallidus, right caudate nucleus, right hippocampus, inferior parietal lobule, left parahippocampal gyrus, and left hippocampus. Notably, there is a predominant occurrence of lesions: granulovacuolar degeneration, Hirano bodies, cotton-wool, and neuritic plaques. The causes of neurodegenerative processes are probably related to TNF-α, IL-1ß, and TGF-ß production and iNOS expression by the NF-κB activation pathway in the R-HP, inducing necroptosis. CONCLUSIONS: The occurrence of neuroinflammatory responses is linked to tissue lesions in AD. The production of inflammatory cytokines is the basis of this process, which ultimately leads to the necroptosis, which is triggered by neuroinflammation amplification. The inhibition of neuroinflammation by targeting TNF-α/iNOS could stop tissue damage, this may be a promising therapeutic pathway.

The involvement of neuroinflammation and necroptosis in the hippocampus during vascular dementia.

The prevalence of vascular dementia is increasing at an alarming rate. The Confirmation of the clinical diagnosis of vascular dementia depends on post-mortem examination of the brain. In our study, we investigated the vascular disease and neuroinflammation during vascular dementia. Our results showed a ß-amyloid deposits, neovascularization, neuronal hypertrophy and neuroinflammation in the hippocampus tissue. Interestingly, the neuroinflammation was characterized by a higher expression of TNF-α, IL-1ß, TGF-ß and iNOS which are TLR4/RelA pathway dependent. Finally, the finding of necroptosis by impaired blood supply and inflammation state suggests that the cognitive impairment was caused by vascular disease and neuroinflammation.

All-trans retinoic acid (ATRA) prevents lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment in aged rats.

We aimed to investigate preventive effects of All-trans retinoic acid (ATRA) on a lipopolysaccharide (LPS)-induced aged neuroinflammation model. We analyzed behavior, systemic nitric oxide (NO) production, cerebral NO synthase (NOS2) and ß-amyloid (Aß) 1-42 expression and tissue integrity in the neuroinflammation model pretreated with ATRA (150µg/ml/rat/day) for 30days. Our results showed that LPS treatment (500µg/kg/day) for 7days disturbed memory, enhanced systemic NO production, NOS2 and Aß 1-42 cerebral expression and generated an Alzheimer's disease (AD)-like neuronal degeneration. Interestingly, ATRA pretreatment prevented the LPS-induced deleterious effects. ATRA could be a potent preventive approach in AD.

IFN-γ and TNF-α are involved during Alzheimer disease progression and correlate with nitric oxide production: a study in Algerian patients.

Alzheimer's disease (AD) is a neurodegenerative disease leading to a progressive and irreversible loss of mental functions. It is characterized by 3 stages according to the evolution and the severity of the symptoms. This disease is associated with an immune disorder, which appears with significant rise in the inflammatory cytokines and increased production of free radicals such as nitric oxide (NO). Our study aims to investigate interferon (IFN)-γ and tumor necrosis factor-α (TNF-α) involvement in NO production, in vivo and ex vivo, in peripheral blood mononuclear cells from Algerian patients (n=25), according to the different stages of the disease (mild Alzheimer's, moderate Alzheimer's, and severe Alzheimer's) in comparison to mild cognitive impairment (MCI) patients. Interestingly, we observed that in vivo IFN-γ and TNF-α levels assessed in patients with AD in mild and severe stages, respectively, are higher than those observed in patients with moderate stage and MCI. Our in vivo and ex vivo results show that NO production is related to the increased levels of IFN-γ and TNF-α, in mild and severe stages of AD. Remarkably, significant IFN-γ level is only detected in mild stage of AD. Our study suggests that NO production is IFN-γ dependent both in MCI and mild Alzheimer's patients. Further, high levels of NO are associated with an elevation of TNF-α levels in severe stage of AD. Collectively, our data indicate that the proinflammatory cytokine production seems, in part, to be involved in neurological deleterious effects observed during the development of AD through NO pathway.

IL-23/IL-17A axis correlates with the nitric oxide pathway in inflammatory bowel disease: immunomodulatory effect of retinoic acid.

Inflammatory bowel diseases (IBDs) are chronic inflammatory diseases of the gastrointestinal tract, which are clinically present as 1 of the 2 disorders, Crohn's disease (CD) or ulcerative colitis (UC) (Rogler 2004). The immune dysregulation in the intestine plays a critical role in the pathogenesis of IBD, involving a wide range of molecules, including cytokines. The aim of this work was to study the involvement of T-helper 17 (Th17) subset in the bowel disease pathogenesis by the nitric oxide (NO) pathway in Algerian patients with IBD. We investigated the correlation between the proinflammatory cytokines [(interleukin (IL)-17, IL-23, and IL-6] and NO production in 2 groups of patients. We analyzed the expression of messenger RNAs (mRNAs) encoding Th17 cytokines, cytokine receptors, and NO synthase 2 (NOS2) in plasma of the patients. In the same way, the expression of p-signal transducer and activator of transcription 3 (STAT3) and NOS2 was measured by immunofluorescence and immunohistochemistry. We also studied NO modulation by proinflammatory cytokines (IL-17A, IL-6, tumor necrosis factor α, or IL-1ß) in the presence or absence of all-trans retinoic acid (At RA) in peripheral blood mononuclear cells (PBMCs), monocytes, and in colonic mucosa cultures. Analysis of cytokines, cytokine receptors, and NOS2 transcripts revealed that the levels of mRNA transcripts of the indicated genes are elevated in all IBD groups. Our study shows a significant positive correlation between the NO and IL-17A, IL-23, and IL-6 levels in plasma of the patients with IBD. Interestingly, the correlation is significantly higher in patients with active CD. Our study shows that both p-STAT3 and inducible NOS expression was upregulated in PBMCs and colonic mucosa, especially in patients with active CD. At RA downregulates NO production in the presence of proinflammatory cytokines for the 2 groups of patients. Collectively, our study indicates that the IL-23/IL-17A axis plays a pivotal role in IBD pathogenesis through the NO pathway.

Involvement of interferon-γ in bowel disease pathogenesis by nitric oxide pathway: a study in Algerian patients.

The pathogenesis of inflammatory bowel disease (IBD) is complex, involving a wide range of molecules including cytokines. Abnormalities in the expression of immunoregulatory cytokines such as interferon-γ (IFN-γ) and interleukin-12 (IL-12) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. The aim of this work was to study the implication of IFN-γ and nitric oxide (NO) in bowel disease pathogenesis. In this study, we investigated the circulating IFN-γ and IL-12 production in 2 groups of Algerian patients with IBD (Crohn's disease and ulcerative colitis). Moreover, systemic NO concentrations and NO generation by colonic mucosa were determined in these patients. Finally, we examined the effect of IFN-γ on NO production by peripheral blood mononuclear cells (PBMCs) of these patients. Our results indicate that IFN-γ/IL-12 production in IBD patients was increased in comparison to healthy donors. This strong production correlates with high levels of NO in sera and colonic mucosa culture. Interestingly, NO production was related to the clinical stage of IBD patients (inactive or active stage). The relationship between IFN-γ and NO production in IBD patients were confirmed by in vitro experiments and the role of IFN-γ in NO synthase induction in patients' PBMC culture was suggested. Collectively, our results show that IFN-γ plays a pivotal role in IBD pathogenesis through NO pathway.