Evaluation of NADPH Oxidase (NOX) Activity by nitro Blue Tetrazolium (NBT) Test in SLE Patients

INTRODUCTION Innate immunity plays a crucial part in initiating and perpetuating the disease process in SLE. Recent studies have pushed the neutrophil, an essential component of the innate immunity, to the forefront of the pathogenesis of SLE.1 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2), constitutively expressed in neutrophils, plays a pivotal role in producing cytosolic reactive oxygen species (ROS).2 ROS-mediated stress has been implicated in various autoimmune and immunodeficiency diseases, including SLE. There is conflicting evidence in the literature on basal and stimulated cytoplasmic. ROS production in SLE patients. Some studies have reported an increased ROS production3 in SLE patients, while others showed a negative association between SLE and ROS.4 Production of ROS depends on neutrophilic NOX2 activity. It is unclear what happens to NOX2 level in SLE and whether it varies in different disease activity states. Recent studies on exacerbation of lupus in NOX2 or its subunit deficient lupus-prone mice5,6 brought forth the idea that SLE patients, particularly those with high disease activity, might have less NOX2 level than usual. Since flares characterize the lupus disease course, the NOX2 levels may be low due to increased disease activity. Measuring the quantity of cytosolic ROS with nitro blue tetrazolium (NBT) test, is an indirect measure of NOX2, as the latter is needed for their production and would therefore give an idea of any correlation between ROS and disease activity in SLE.


INTRODUCTION
Innate immunity plays a crucial part in initiating and perpetuating the disease process in SLE.Recent studies have pushed the neutrophil, an essential component of the innate immunity, to the forefront of the pathogenesis of SLE. 1 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2), constitutively expressed in neutrophils, plays a pivotal role in producing cytosolic reactive oxygen species (ROS). 2 ROS-mediated stress has been implicated in various autoimmune and immunodeficiency diseases, including SLE.There is conflicting evidence in the literature on basal and stimulated cytoplasmic.ROS production in SLE patients.Some studies have reported an increased ROS production 3 in SLE patients, while others showed a negative association between SLE and ROS. 4 Production of ROS depends on neutrophilic NOX2 activity.It is unclear what happens to NOX2 level in SLE and whether it varies in different disease activity states.Recent studies on exacerbation of lupus in NOX2 or its subunit deficient lupus-prone mice 5,6 brought forth the idea that SLE patients, particularly those with high disease activity, might have less NOX2 level than usual.Since flares characterize the lupus disease course, the NOX2 levels may be low due to increased disease activity.
Measuring the quantity of cytosolic ROS with nitro blue tetrazolium (NBT) test, is an indirect measure of NOX2, as the latter is needed for their production and would therefore give an idea of any correlation between ROS and disease activity in SLE.

OBJECTIVE
The primary objective of the study was to compare cytosolic reactive oxygen species (ROS) production (both basal and after stimulation with PMA) using Nitro blue tetrazolium (NBT) assay in high versus low disease activity states in SLE patients and healthy controls.Other objectives were to compare the level of ROS production with disease activity, clinical and serological status and different outcome measures -SLE disease activity index-2K (SLEDAI-2K), Physician global assessment by visual analogue scale (PhGA -VAS), British Isles Lupus assessment group index (BILAG scores).

Study Population
This was a cross-

Isolation of Neutrophils from peripheral blood
A volume of 6 ml of peripheral blood was collected from subjects in an EDTA vial and immediately sent for processing.The sample was diluted with 1x PBS (pH 7.4) buffer without calcium & magnesium ions at a 1:1 ratio.Diluted blood was layered over 4-5 ml of Ficoll-paque solution and centrifuged at 2000 rpm for 20 min at room temperature.After centrifugation, distinct layers were observed, with plasma, peripheral blood mononuclear cells (PBMC), Lyphodex solution, and pellets of sedimented red blood cells (RBC) with granulocytes.The first three layers of plasma, PBMC and Lyphodex solution were discarded to obtain RBC pellets with granulocytes.The RBC pellets were immediately suspended in 2 ml of 1X PBS buffer for further processing.Dextran sedimentation was performed with a 6% dextran solution to separate the neutrophils from RBC sediment.Neutrophil-rich supernatant was collected and centrifuged for 10 minutes.Then RBC lysis was done to remove residual RBCs as supernatant and a white pellet consisting of granulocytes was obtained.

Morphology verification
After lysis, morphological examination and trypan blue exclusion test were performed to determine cell count and purity of neutrophils.Freshly isolated neutrophils were stained with 0.4% trypan blue and mixed under countess™ automated cell counter.The morphological observation revealed that >95% of cells isolated were live neutrophils.Analysis of neutrophil purity was performed by flow cytometry.(Supplementary Figure 1s)

NBT reduction
The Nitro blue tetrazolium (NBT) 0.1% solution was prepared by adding 1mg of NBT powder (Sigma) to 10mL of PBS (pH 7.2) and filtered with a 0.2mm filter.NBT assay was performed using freshly isolated neutrophils (1x10

Demographic characteristics
The total number of SLE patients and controls included were 73 and 46, with a mean age of 28.  1).

NBT reduction according to disease activity
The basal NBT reduction was significantly higher in cases with high disease activity than those with low disease activity (0.46 ± 0.29 vs 0.31 ± 0.11, p=0.015).Stimulated NBT reduction in patients with high disease activity compared to low disease activity was not significantly different.Kendall-Tau correlation matrix showed the SLEDAI-2K was strongly associated with higher basal and ∆ROS (increment in ROS) production (Figure 2).However, levels of dsDNA or complements did not show any significant association.Patients with lupus nephritis showed higher basal NBT reduction than without nephritis (0.43±0.26 vs 0.30±0.09,p=0.0001) (Table 3), but there was no statistically significant difference between individual renal BILAG domains (Figure 3).PhGA-VAS scale also showed a significant correlation with both basal and stimulated ROS production (p=0.04 & 0.003, respectively).The association of ROS with demographic and clinical  4) Kendall-Tau correlation matrix also showed the basal and ∆ROS (increment in ROS) levels were not influenced by age, gender, total leukocyte count, neutrophil count, lymphocyte count, haemoglobin, and platelet count.However, high steroid dose increased basal ROS and ∆ROS values while comorbidities decreased the basal and ∆ROS.(Supplementary Figure 4s)

DISCUSSION
In the present study, using the NBT test, we showed that SLE subjects had higher stimulated cytosolic ROS production than healthy controls.Basal ROS generation significantly increased in high disease activity state compared to low disease activity.In patients with lupus nephritis, there was a significantly higher basal ROS level.Age, gender, comorbidities, high steroid dose, other medications, leukopenia, dsDNA, and past infections explain one-fifth of the increase in ROS with stimulation.Perazzio et al. 3 have reported increased oxidative burst in SLE patients both at basal and stimulated state compared to healthy controls.Our study confirms their observation.SLE neutrophils may be primed for respiratory burst due to the existing inflammatory milieu.Though several groups have previously shown increased ROS production by SLE neutrophils after stimulation, [9][10][11][12][13] there is contradictory evidence in the literature regarding the oxidative status of SLE neutrophils upon stimulation.High dose steroid: >-10 mg prednisolone or equivalent/day.Other medications include any steroid sparing immunosuppressive -azathioprine, cyclophosphamide, mycophenolate, etc.The matrix represents correlation coefficients of pairwise variables.
The red background depicts inverse association, while the green background depicts positive association.

TITLE
Bengtsson et al. 4 reported low respiratory burst using Phago-Burst assay and 2,7-dichlorofluorescein-diacetate (DCFH-DA) test on a cohort of Swedish SLE patients compared to controls.However, this cohort of patients had lower disease activity, fewer patients with nephritis, and a greater proportion of patients with organ damage.These factors could explain the low respiratory burst observed.Marzocchi et al. 14 have also reported a significant decrease in the oxidative burst of neutrophils in Brazilian patients with active SLE than controls.Here they examined the complement and FcϒR mediated oxidative burst rather than PMA stimulated oxidative burst.It was found that FcϒR levels were depleted in the inflammatory milieu, and this assessed a pathway of ROS generation different from our study.In another study by Philip et al., 15 using the NBT test, no difference in the oxidative burst was demonstrated between SLE cases and healthy controls.A few studies have reported a negative association between SLE and ROS generation.The present study is in conformation with most of the previous studies proving a connection between SLE and ROS production.Perazzio et al. did not find any correlation between disease activity and oxidative burst in SLE neutrophils.This finding led them to suggest that changes in ROS levels are independent of the inflammatory process.Instead, it is influenced by the ongoing subclinical immunological disorder present even in patients with apparently quiescent disease.Our results differed from the above conclusions.We have demonstrated that basal cytosolic ROS production was higher with increased disease activity with a strong positive correlation between SLEDAI and increment in ROS production on stimulation.Though in several studies, it was found that high disease activity was associated with an increased oxidative burst, 16,17 the exact association between SLE activity and neutrophilic ROS generation is still unclear.Widespread tissue inflammatory processes could probably cause a more significant amount of ROS production even at the basal state by the neutrophils in patients with active SLE.There is no data available in literature where neutrophil oxidase activity was correlated with the BILAG score.In this study, no difference was found in basal and stimulated ROS production with BILAG scoring of mucocutaneous, renal, and haematological domains.
Our study demonstrates that lupus nephritis patients had a significantly higher ROS production in the basal state than those without nephritis.Production of ROS on stimulation is also greater in this group, even though it was not statistically significant.This finding was similar to Perazzio et al., where patients with lupus nephritis presented higher ROS production increments after stimulus with S. aureus than those without nephritis.But Phillip et al. did not find any association between nephritis and basal ROS production.

Figure 3 .
Figure 3.Comparison of basal, stimulated NBT reduction for each category (A, B, C, D, E) of renal BILAG domain.

Table 1 .
Demographic and clinical characteristics of the studied SLE patients and controls.
Statistical analysisMean value of basal, stimulated, and change in ROS production as measured by NBT were compared between SLE cases and healthy control with student unpaired t-test.Kendall-Tau correlation matrix was performed to detect the influence of disease activity on basal, stimulated, and change in ROS production.Pooled analysis of basal, TITLE stimulated, and change in ROS production was done for different groups, including case and healthy controls.Basal and stimulated ROS levels were compared in each category (A, B, C, D, E) of BILAG renal, mucocutaneous, and hematologic domains by ANOVA test.IBM SPSS 2021 version was used for statistical analysis.

Table 2 .
Basal and stimulated NBT reduction (mean ± SD) in case versus control.

Table 3 .
Basal and stimulated NBT reduction (mean ± SD) in lupus nephritis patients.

Table 4 .
Multiple Linear Regression -Ordinary Least Squares showing association of ∆NBT values with demographic and clinical variables.