Oxidative Stress-Related Mechanisms in SARS-CoV-2 Infections.

The COVID-19 pandemic caused relatively high mortality in patients, especially in those with concomitant diseases (i.e., diabetes, hypertension, and chronic obstructive pulmonary disease (COPD)). In most of aforementioned comorbidities, the oxidative stress appears to be an important player in their pathogenesis. The direct cause of death in critically ill patients with COVID-19 is still far from being elucidated. Although some preliminary data suggests that the lung vasculature injury and the loss of the functioning part of pulmonary alveolar population are crucial, the precise mechanism is still unclear. On the other hand, at least two classes of medications used with some clinical benefits in COVID-19 treatment seem to have a major influence on ROS (reactive oxygen species) and RNS (reactive nitrogen species) production. However, oxidative stress is one of the important mechanisms in the antiviral immune response and innate immunity. Therefore, it would be of interest to summarize the data regarding the oxidative stress in severe COVID-19. In this review, we discuss the role of oxidative and antioxidant mechanisms in severe COVID-19 based on available studies. We also present the role of ROS and RNS in other viral infections in humans and in animal models. Although reactive oxygen and nitrogen species play an important role in the innate antiviral immune response, in some situations, they might have a deleterious effect, e.g., in some coronaviral infections. The understanding of the redox mechanisms in severe COVID-19 disease may have an impact on its treatment.

Cold Atmospheric Plasma Promotes the Immunoreactivity of Granulocytes In Vitro.

Cold atmospheric plasma (CAP) reduces bacteria and interacts with tissues and cells, thus improving wound healing. The CAP-related induction of neutrophils was recently described in stained sections of wound tissue in mice. Consequently, this study aimed to examine the functionality of human polymorphonuclear cells (PMN)/granulocytes through either a plasma-treated solution (PTS) or the direct CAP treatment with different plasma modes and treatment durations. PTS analysis yielded mode-dependent differences in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) after CAP treatment. Live-cell imaging did not show any chemo-attractive or NETosis-inducing effect on PMNs treated with PTS. The time to maximum ROS production (TmaxROS) in PMNs was reduced by PTS and direct CAP treatment. PMNs directly treated with CAP showed an altered cell migration dependent on the treatment duration as well as decreased TmaxROS without inducing apoptosis. Additionally, flow cytometry showed enhanced integrin and selectin expression, as a marker of activation, on PMN surfaces. In conclusion, the modification of PMN immunoreactivity may be a main supporting mechanism for CAP-induced improvement in wound healing.

Construction of a nitro-oxidative stress-driven, mechanistic model of mood disorders: A nomothetic network approach.

Major depression is accompanied by increased IgM-mediated autoimmune responses to oxidative specific epitopes (OSEs) and nitric oxide (NO)-adducts. These responses were not examined in bipolar disorder type 1 (BP1) and BP2. IgM responses to malondialdehyde (MDA), phosphatidinylinositol, oleic acid, azelaic acid, and NO-adducts were determined in 35 healthy controls, and 47 major depressed (MDD), 29 BP1, and 25 BP2 patients. We also measured serum peroxides, IgG to oxidized LDL (oxLDL), and IgM/IgA directed to lipopolysaccharides (LPS). IgM responses to OSEs and NO-adducts (OSENO) were significantly higher in MDD and BP1 as compared with controls, and IgM to OSEs higher in MDD than in BP2. Partial Least Squares (PLS) analysis showed that 57.7% of the variance in the clinical phenome of mood disorders was explained by number of episodes, a latent vector extracted from IgM to OSENO, IgG to oxLDL, and peroxides. There were significant specific indirect effects of IgA/IgM to LPS on the clinical phenome, which were mediated by peroxides, IgM OSENO, and IgG oxLDL. Using PLS we have constructed a data-driven nomothetic network which ensembled causome (increased plasma LPS load), adverse outcome pathways (namely neuro-affective toxicity), and clinical phenome features of mood disorders in a data-driven model. Based on those feature sets, cluster analysis discovered a new diagnostic class characterized by increased plasma LPS load, peroxides, autoimmune responses to OSENO, and increased phenome scores. Using the new nomothetic network approach, we constructed a mechanistically transdiagnostic diagnostic class indicating neuro-affective toxicity in 74.3% of the mood disorder patients.

Reactive oxygen and nitrogen species and innate immune response.

The innate immune system is the first line of defense against pathogens and is characterized by its fast but nonspecific response. One important mechanism of this system is the production of the biocidal reactive oxygen and nitrogen species, which are widely distributed within biological systems, including phagocytes and secretions. Reactive oxygen and nitrogen species are short-lived intermediates that are biochemically synthesized by various enzymatic reactions in aerobic organisms and are regulated by antioxidants. The physiological levels of reactive species play important roles in cellular signaling and proliferation. However, higher concentrations and prolonged exposure can fight infections by damaging important microbial biomolecules. One feature of the reactive species generation system is the interaction between its components to produce more biocidal agents. For example, the phagocytic NADPH oxidase complex generates superoxide, which functions as a precursor for antimicrobial hydrogen peroxide synthesis. Peroxide is then used by myeloperoxidase in the same cells to generate hypochlorous acid, a highly microbicidal agent. Studies on animal models and microorganisms have shown that deficiency of these antimicrobial agents is associated with severe recurrent infections and immunocompromised diseases, such as chronic granulomatous disease. There is accumulating evidence that reactive species have important positive aspects on human health and immunity; however, some important promising features of this system remain obscure.

Temporal Dynamics of Reactive Oxygen and Nitrogen Species and NF-κB Activation During Acute and Chronic T Cell-Driven Inflammation.

PURPOSE: Reactive oxygen and nitrogen species (ROS/RNS) production and the NF-κB activation are critically involved in inflammatory responses, but knowledge about the temporal dynamics during acute and chronic inflammation is limited. Here, we present a comparative longitudinal in vivo study of both parameters in an experimental model of acute and chronic T cell-driven delayed-type hypersensitivity reaction (DTHR) using noninvasive optical imaging. PROCEDURES: Trinitrochlorobenzene (TNCB)-sensitized NF-κB-luciferase-reporter and wild-type mice were TNCB challenged on the right ear to elicit acute DTHR and then repetitively challenged (up to five times) to induce chronic DTHR. Mice were treated with the ROS-scavenging and NF-κB inhibiting molecule N-acetylcysteine (NAC) or underwent sham treatment. ROS/RNS production was noninvasively analyzed in vivo using the ROS-/RNS-sensitive chemiluminescent probe L-012, and NF-κB activation was measured using NF-κB-luciferase-reporter mice. H&E staining, CD3 and myeloperoxidase (MPO) immunohistochemistry (IHC), and quantitative PCR (qPCR) analyses were employed to investigate immune cell infiltration and expression of NF-κB- and ROS-/RNS-driven genes. RESULTS: In acute DTHR, we found strongly elevated ROS/RNS production and NF-κB activation 12 h after the 1st TNCB ear challenge, peaking at 24 h after the challenge. In chronic DTHR, ROS production peaked as early as 4 h after the 5th TNCB challenge, whereas NF-κB activity peaked after 12 h. The increase in ROS/RNS production in acute DTHR was higher than the increase in NF-κB activity but the relationship was inverse in chronic DTHR. Treatment with the ROS scavenger NAC had differential effects on ROS/RNS production and NF-κB activation during acute and chronic DTHR. Ex vivo cross-validation by histopathology and qPCR analysis correlated closely with the in vivo imaging results. CONCLUSIONS: Noninvasive in vivo imaging is capable of assessing the temporal dynamics of ROS/RNS production and NF-κB activation during progression from acute to chronic DTHR and enables monitoring of anti-inflammatory treatment responses.

Regulation of T Cell Function by Reactive Nitrogen and Oxygen Species in Collagen-Induced Arthritis.

Aims: In this study, we investigate the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in autoimmune diseases. We focus on oxidative regulation at the interaction between antigen-presenting cells (APCs) and T cells, and consequent effect of ROS and RNS on type II collagen (CII)-induced arthritis (CIA) model in mice. Results: Mice deficient in ROS and peroxide, due to a mutation in Ncf1 gene, develop an exaggerated CIA and a stronger T cell response to CII. In contrast, nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was found to protect against CIA. The most pronounced protective effect was observed when L-NAME treatment started immediately after CII immunization. Ten days after immunization, the CII-reactive T cell-proliferative response was greater in Ncf1-mutant mice that were treated with L-NAME. T cells from L-NAME-treated mice, primed with CII, showed lower interleukin-2 secretion in response to CII in vitro. Moreover, inhibition of RNS production resulted in dysregulation of NOS1 (neuronal) expression in CII-reactive T cells. Innovation and Conclusion: The results support that deficiency of a paracrine factor as ROS and peroxide released by APC leads to pronounced activation of T cells and enhanced arthritis. An intrinsic factor might be RNS produced by NOS1, which likely enhanced T cell activation in an autocrine manner.

Staphylococcus aureus Lipoic Acid Synthesis Limits Macrophage Reactive Oxygen and Nitrogen Species Production To Promote Survival during Infection.

Macrophages are critical mediators of innate immunity and must be overcome for bacterial pathogens to cause disease. The Gram-positive bacterium Staphylococcus aureus produces virulence factors that impede macrophages and other immune cells. We previously determined that production of the metabolic cofactor lipoic acid by the lipoic acid synthetase, LipA, blunts macrophage activation. A ΔlipA mutant was attenuated during infection and was more readily cleared from the host. We hypothesized that bacterial lipoic acid synthesis perturbs macrophage antimicrobial functions and therefore hinders the clearance of S. aureus Here, we found that enhanced innate immune cell activation after infection with a ΔlipA mutant was central to attenuation in vivo, whereas a growth defect imparted by the lipA mutation made a negligible contribution to overall clearance. Macrophages recruited to the site of infection with the ΔlipA mutant produced larger amounts of bactericidal reactive oxygen species (ROS) and reactive nitrogen species (RNS) than those recruited to the site of infection with the wild-type strain or the mutant strain complemented with lipA ROS derived from the NADPH phagocyte oxidase complex and RNS derived from the inducible nitric oxide synthetase, but not mitochondrial ROS, were critical for the restriction of bacterial growth under these conditions. Despite enhanced antimicrobial immunity upon primary infection with the ΔlipA mutant, we found that the host failed to mount an improved recall response to secondary infection. Our data suggest that lipoic acid synthesis in S. aureus promotes bacterial persistence during infection through limitation of ROS and RNS generation by macrophages. Broadly, this work furthers our understanding of the intersections between bacterial metabolism and immune responses to infection.

Programmed necrotic cell death of macrophages: Focus on pyroptosis, necroptosis, and parthanatos.

Macrophages are highly plastic cells of the innate immune system. Macrophages play central roles in immunity against microbes and contribute to a wide array of pathologies. The processes of macrophage activation and their functions have attracted considerable attention from life scientists. Although macrophages are highly resistant to many toxic stimuli, including oxidative stress, macrophage death has been reported in certain diseases, such as viral infections, tuberculosis, atherosclerotic plaque development, inflammation, and sepsis. While most studies on macrophage death focused on apoptosis, a significant body of data indicates that programmed necrotic cell death forms may be equally important modes of macrophage death. Three such regulated necrotic cell death modalities in macrophages contribute to different pathologies, including necroptosis, pyroptosis, and parthanatos. Various reactive oxygen and nitrogen species, such as superoxide, hydrogen peroxide, and peroxynitrite have been shown to act as triggers, mediators, or modulators in regulated necrotic cell death pathways. Here we discuss recent advances in necroptosis, pyroptosis, and parthanatos, with a strong focus on the role of redox homeostasis in the regulation of these events.

Pyridinecarboxylic Acid Derivative Stimulates Pro-Angiogenic Mediators by PI3K/AKT/mTOR and Inhibits Reactive Nitrogen and Oxygen Species and NF-κB Activation Through a PPARγ-Dependent Pathway in T. cruzi-Infected Macrophages.

Chagas disease is caused by Trypanosoma cruzi infection and represents an important public health concern in Latin America. Macrophages are one of the main infiltrating leukocytes in response to infection. Parasite persistence could trigger a sustained activation of these cells, contributing to the damage observed in this pathology, particularly in the heart. HP24, a pyridinecarboxylic acid derivative, is a new PPARγ ligand that exerts anti-inflammatory and pro-angiogenic effects. The aim of this work was to deepen the study of the mechanisms involved in the pro-angiogenic and anti-inflammatory effects of HP24 in T. cruzi-infected macrophages, which have not yet been elucidated. We show for the first time that HP24 increases expression of VEGF-A and eNOS through PI3K/AKT/mTOR and PPARγ pathways and that HP24 inhibits iNOS expression and NO release, a pro-inflammatory mediator, through PPARγ-dependent mechanisms. Furthermore, this study shows that HP24 modulates H2O2 production in a PPARγ-dependent manner. It is also demonstrated that this new PPARγ ligand inhibits the NF-κB pathway. HP24 inhibits IKK phosphorylation and IκB-α degradation, as well as p65 translocation to the nucleus in a PPARγ-dependent manner. In Chagas disease, both the sustained increment in pro-inflammatory mediators and microvascular abnormalities are crucial aspects for the generation of cardiac damage. Elucidating the mechanism of action of new PPARγ ligands is highly attractive, given the fact that it can be used as an adjuvant therapy, particularly in the case of Chagas disease in which inflammation and tissue remodeling play an important role in the pathophysiology of this disease.

The bacterial pigment pyocyanin inhibits the NLRP3 inflammasome through intracellular reactive oxygen and nitrogen species.

Inflammasomes are cytosolic complexes that mature and secrete the inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18 and induce pyroptosis. The NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome detects many pathogen- and danger-associated molecular patterns, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) have been implicated in its activation. The phenazine pyocyanin (PCN) is a virulence factor of Pseudomonas aeruginosa and generates superoxide in cells. Here we report that PCN inhibits IL-1ß and IL-18 release and pyroptosis upon NLRP3 inflammasome activation in macrophages by preventing speck formation and Caspase-1 maturation. Of note, PCN did not regulate the AIM2 (absent in melanoma 2) or NLRC4 inflammasomes or tumor necrosis factor (TNF) secretion. Imaging of the fluorescent glutathione redox potential sensor Grx1-roGFP2 indicated that PCN provokes cytosolic and nuclear but not mitochondrial redox changes. PCN-induced intracellular ROS/RNS inhibited the NLRP3 inflammasome posttranslationally, and hydrogen peroxide or peroxynitrite alone were sufficient to block its activation. We propose that cytosolic ROS/RNS inhibit the NLRP3 inflammasome and that PCN's anti-inflammatory activity may help P. aeruginosa evade immune recognition.

Redox regulation of cardiovascular inflammation - Immunomodulatory function of mitochondrial and Nox-derived reactive oxygen and nitrogen species.

Oxidative stress is a major hallmark of cardiovascular diseases although a causal link was so far not proven by large clinical trials. However, there is a close association between oxidative stress and inflammation and increasing evidence for a causal role of (low-grade) inflammation for the onset and progression of cardiovascular diseases, which may serve as the missing link between oxidative stress and cardiovascular morbidity and mortality. With the present review we would like to highlight the multiple redox regulated pathways in inflammation, discuss the sources of reactive oxygen and nitrogen species that are of interest for these processes and finally discuss the importance of angiotensin II (AT-II) as a trigger of cardiovascular inflammation and the initiation and progression of cardiovascular diseases.

TNF and ROS Crosstalk in Inflammation.

Tumor necrosis factor (TNF) is tremendously important for mammalian immunity and cellular homeostasis. The role of TNF as a master regulator in balancing cell survival, apoptosis and necroptosis has been extensively studied in various cell types and tissues. Although these findings have revealed much about the direct impact of TNF on the regulation of NF-κB and JNK, there is now rising interest in understanding the emerging function of TNF as a regulator of the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In this review we summarize work aimed at defining the role of TNF in the control of ROS/RNS signaling that influences innate immune cells under both physiological and inflammatory conditions.

iNOS null MRL+/+ mice show attenuation of trichloroethene-mediated autoimmunity: contribution of reactive nitrogen species and lipid-derived reactive aldehydes.

Earlier studies from our laboratory in MRL+/+ mice suggest that free radicals, especially overproduction of reactive nitrogen species (RNS) and lipid-derived reactive aldehydes (LDRAs), are associated with trichloroethene (TCE)-mediated autoimmune response. The current study was undertaken to further assess the contribution of RNS and LDRAs in TCE-mediated autoimmunity by using iNOS-null MRL+/+ mice. iNOS-null MRL+/+ mice were obtained by backcrossing iNOS-null mice (B6.129P2-Nos2(tm1Lau)/J) to MRL +/+ mice. Female MRL+/+ and iNOS-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4(th) day) for 6 weeks; their respective controls received corn oil only. TCE exposure led to significantly increased iNOS mRNA in livers, iNOS protein in livers and sera, increased nitrotyrosine (NT) formation in both livers and sera, induction of MDA-/HNE-protein adducts in livers and their respective antibodies in sera along with significant increases in serum antinuclear antibodies (ANA) and anti-dsDNA in MRL+/+ mice. Even though in iNOS-null MRL+/+ mice, the iNOS and NT levels were negligible in both TCE-treated and untreated groups, TCE treatment still led to significant increases in MDA-/HNE-protein adducts and their respective antibodies along with increases in serum ANA and anti-dsDNA compared to controls. Most remarkably, the increases in serum ANA and anti-dsDNA induced by TCE in the iNOS-null MRL+/+ mice were significantly less pronounced compared to that in MRL+/+ mice. Our results provide further evidence that both RNS and LDRAs contribute to TCE-induced autoimmunity in MRL+/+ mice, and iNOS deficiency attenuates this autoimmune response.

Glucocorticoid-Augmented Efferocytosis Inhibits Pulmonary Pneumococcal Clearance in Mice by Reducing Alveolar Macrophage Bactericidal Function.

Inhaled corticosteroids (ICS) increase community-acquired pneumonia (CAP) incidence in patients with chronic obstructive pulmonary disease (COPD) by unknown mechanisms. Apoptosis is increased in the lungs of COPD patients. Uptake of apoptotic cells (ACs) ("efferocytosis") by alveolar macrophages (AMøs) reduces their ability to combat microbes, including Streptococcus pneumoniae, the most common cause of CAP in COPD patients. Having shown that ICS significantly increase AMø efferocytosis, we hypothesized that this process, termed glucocorticoid-augmented efferocytosis, might explain the association of CAP with ICS therapy in COPD. To test this hypothesis, we studied the effects of fluticasone, AC, or both on AMøs of C57BL/6 mice in vitro and in an established model of pneumococcal pneumonia. Fluticasone plus AC significantly reduced TLR4-stimulated AMø IL-12 production, relative to either treatment alone, and decreased TNF-α, CCL3, CCL5, and keratinocyte-derived chemoattractant/CXCL1, relative to AC. Mice treated with fluticasone plus AC before infection with viable pneumococci developed significantly more lung CFUs at 48 h. However, none of the pretreatments altered inflammatory cell recruitment to the lungs at 48 h postinfection, and fluticasone plus AC less markedly reduced in vitro mediator production to heat-killed pneumococci. Fluticasone plus AC significantly reduced in vitro AMø killing of pneumococci, relative to other conditions, in part by delaying phagolysosome acidification without affecting production of reactive oxygen or nitrogen species. These results support glucocorticoid-augmented efferocytosis as a potential explanation for the epidemiological association of ICS therapy of COPD patients with increased risk for CAP, and establish murine experimental models to dissect underlying molecular mechanisms.

Reactive oxygen species in psoriasis and psoriasis arthritis: relevance to human disease.

Psoriasis (Ps) is a chronic, immune-mediated, skin inflammatory disease affecting up to 3% of the population worldwide. Different environmental triggers initiate this complex multifactorial syndrome. Many individuals affected by Ps (6-26%) develop inflammatory disease in other organs, often in the joints as in psoriasis arthritis (PsA). Animal models that reflect the typical Ps syndrome, including both skin and joint pathology as in Ps and PsA, are valuable tools for dissecting disease pathways leading to clinical manifestations. In this context, we developed a new acute Ps and PsA-like disease model that appears after exposure to Saccharomyces cerevisiae mannan in certain mouse strains. The disease was found to be triggered by mannan-activated macrophages, leading to the activation of a pathogenic interleukin-17 pathway involving innate lymphocytes. Interestingly, the production of reactive oxygen species protected the mice from the triggering of this pathway and ameliorated Ps and PsA development.

Neutrophils: The Role of Oxidative and Nitrosative Stress in Health and Disease.

Neutrophils constitute the first line of the innate immunity in humans. They employ several strategies to trap and kill microorganisms, such as phagocytosis, degranulation, and the formation of extracellular traps (NETs). It has been well documented, that generation of reactive oxygen and nitrogen species (ROS and RNS) is crucial in the life cycle of a polymorphonuclear phagocyte. These compounds due to high reactivity act as powerful antimicrobial factors in the process of pathogens clearance and can also modulate immunological response. On the other hand, excessive amount of free radicals may have detrimental effect on host tissues and markers of oxidative and nitrosative stress are detectable in many diseases. It is necessary to maintain the balance between ROS/RNS formation and removal. The review highlights our current understanding of the role of ROS and RNS produced by neutrophils in health and disease.

The response of Paracoccidioides spp. to nitrosative stress.

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.

Limiting immunopathology: Interaction between carotenoids and enzymatic antioxidant defences.

The release of reactive oxygen and nitrogen species (ROS and RNS) during the inflammatory response generates damages to host tissues, referred to as immunopathology, and is an important factor in ecological immunology. The integrated antioxidant system, comprising endogenous antioxidant enzymes (e.g. superoxide dismutase SOD, and catalase CAT) and dietary antioxidants (e.g. carotenoids), helps to cope with immune-mediated oxidative stress. Crustaceans store large amounts of dietary carotenoids for yet unclear reasons. While being immunostimulants and antioxidants, the interaction of these pigments with antioxidant enzymes remains unclear. Here, we tested the interaction between dietary supplementation with carotenoids and immune challenge on immune defences and the activity of the antioxidant enzymes SOD and CAT, in the amphipod crustacean Gammarus pulex. Dietary supplementation increased the concentrations of circulating carotenoids and haemocytes in the haemolymph, while the immune response induced the consumption of circulating carotenoids and a drop of haemocyte density. Interestingly, supplemented gammarids exhibited down-regulated SOD activity but high CAT activity compared to control ones. Our study reveals specific interactions of dietary carotenoids with endogenous antioxidant enzymes, and further underlines the potential importance of carotenoids in the evolution of immunity and/or of antioxidant mechanisms in crustaceans.

[The effect of Xinfeng capsule treatment on the number of BTLA(+)T cells and oxidative stress of patients with ankylosing spondylitis].

OBJECTIVE: To investigate the changes of B and T lymphocyte attenuator (BTLA), reactive oxygen species (ROS), reactive nitrogen species (RNS), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), total antioxidative capacity (TAOC) in the patients with ankylosing spondylitis (AS) and the effect of Xinfeng capsule (XFC) on them. METHODS: AS patients (n=140) were randomly divided into two groups, XFC group (3 tablets each time, tid, n=70) and salicylazosulfapyridine (SASP) group (4 pills each time, bid, n=70). Continuous treatment lasts 3 months. The study also enrolled 60 healthy volunteers as a control group. Flow cytometry was used to test BTLA expression. ELISA was performed to detect the oxidative stress indicators (ROS, RNS, MDA, SOD, CAT, TAOC) and cytokines (IL-4, IL-10, IL-1ß, TNF-α). Western blotting was adopted to examine the blood sedimentation (ESR). HITACHI 7060 automatic biochemical analyzer was used to determine the level of high sensitive C-reactive protein (Hs-CRP). RESULTS: Clinical efficacy of XFC group was significantly better than that of SASP group (P<0.01). Compared with the healthy control group, AS patients had significantly lower BTLA expression in CD3(+) T cells and CD4(+) T cells from the peripheral blood (P<0.01 or P<0.05), the decreased levels of SOD, CAT and TAOC, and significantly increased ROS, RNS and MDA values (P<0.01 or P<0.05). In addition, the levels of serum IL-1ß, TNF-α, ESR and Hs-CRP were significantly higher (P<0.01) and IL-4, IL-10 were significantly lower in AS patients (P<0.01 or P<0.05). Compared with pre-treatment, both XFC and SASP significantly elevated the expressions of BTLA(+)CD3(+) T, BTLA(+)CD4(+) T, BTLA, SOD, TAOC, IL-4, SF-36 (PF, SF, RP, RE, BP, MH, VT, GH) eight dimension scores, and reduced ROS, MDA, TNF-α, ESR, Hs-CRP, VAS, BASDAI, BASFI and BAS-G in the peripheral blood (P<0.01 or P<0.05). The differences between XFC group and SASP group were statistically significant (P<0.01 or P<0.05). Pearson correlation analysis showed that BTLA expression level in the peripheral blood was positively correlated with SOD, RP, BP, SF and RE. BTLA(+)CD3(+) T cells and BTLA*CD4(+) T cells were significantly negatively correlated with ROS, MDA, IL-1ß, TNF-α, ESR, VAS and BASDAI, and they were positively correlated with TAOC, IL-4 and IL-10. BTLA(+)CD3(+) T cells were significantly negatively correlated with RNS, Hs-CRP and BASFI; BTLA(+)CD4(+) T cells were positively correlated with CAT. CONCLUSION: XFC can improve BTLA expression in the peripheral blood of AS patients and regulate negatively the activation and proliferation of T cells.