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Abstract
Background: Activation of the NLRC4 inflammasome appears to start many signalling processes inside the host, including caspase-1, the principal protease responsible for converting proIL-1β and IL-18 to active, secreted IL-1β and IL-18, resulting in pyroptosis. Aims: To evaluate NLRC4 level in patient's blood serum to highlight its role in the pathogenesis of leprosy. Materials and Methods: This prospective study was conducted on 40 patients with leprosy and 30 healthy individuals of matched ages and sexes. All patients were subjected to complete history taking, general and dermatological examination, laboratory investigations, slit skin smear with bacillary index, and clinical classification of the studied leprosy group patients regarding disability according to disability grading. And finally, measurement of serum NLRC4 level by ELISA. Results: In the paucibacillary (PB) group, NLRC4 serum level ranged from 0.9 to 1.8 ng/ml with 1.43 ± 0.28 ng/ml, while in the multibacillary (MB) group, it ranged from 1.2 to 5.7 ng/ml with 2.83 ± 1.11 ng/ml. NLRC4 serum level had increased significantly in MB patients compared to PB patients (P < 0.05). There was a significant difference among the three studied groups, regarding the serum level of NLRC4 (P < 0.05). In leprosy patients, significant positive correlations were found between serum levels of NLRC4 and bacillary index and duration of leprosy. Conclusions: Leprosy patients had considerably greater serum levels of NLRC4 than controls. It was much greater in MB patients than in PB patients.
Keywords: Inflammasome, leprosy, nerve affection, NLRC4
Introduction 
Mycobacterium leprae (M.leprae) is responsible for leprosy, a chronic infectious disease. The primary cause of its pathophysiology is the bacterial invasion of Schwann cells and macrophages in the skin, which leads to tissue death. The condition affects the skin and peripheral nerves and causes irreparable harm if identified late or left untreated.[1]
Ridley and Jopling's categorisation splits leprosy into two polar forms: tuberculoid (TT) and lepromatous (LL). The immunologically unstable intermediate group is divided into three subgroups: borderline tuberculosis (BT), borderline borderline (BB), and borderline leprosy (BL). This classification is determined by clinical criteria, the bacillary index of a skin smear, and histological characteristics.[2][Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
Figure 1: 40-year-old male patient with TT presented with single dry, erythematous, and scaly plaque with loss of hair in the left arm since 6 months
Figure 2: 35-year-old male patient with TT presented with single dry, erythematous, and scaly plaque with loss of hair in the left arm since 4 months
Figure 3: 12-year-old teenager with TT presented with single well defined dry and scaly patch with loss of hair in the left thigh since 6 months
Figure 4: 19-years-old female with LL presented with multiple erythematous papules and nodules in upper limbs since 1 year
Figure 5: 45-year-old male patient with LL presented with multiple nodules in both upper limbs and abdomen since 1 year
Figure 6: 50-year-old male patient with borderline leprosy presented with an erythematous infiltrated plaque since 10 monthsOn the basis of WHO standards, leprosy patients are divided into two types: Paucibacillary (PB), which includes tuberculoid leprosy and some borderline tuberculoid leprosy, and Multibacillary (MB), which includes a borderline group and lepromatous leprosy. On the one hand, TT is characterised by low or non-existent specific antibodies and cell-mediated immunity (CMI) of the Th1 type. LL is characterised by Th2-type immune responses, high antibody production, and limited or absent M.leprae-specific CMI.[3]
The identification of M. leprae by the host's innate immune system is the first step in combating the invading bacteria and is essential for the initiation of the adaptive immune response to infection. The inflammasome is a protein complex in the cytosol that mediates the inflammatory response to pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively). The latter is in charge of the maturation of caspase 1, the release of IL-1β and IL-18, and pyroptosis, a kind of planned cell death. Pattern recognition receptors (PRRs), an adapter protein, and an effector enzyme constitute inflammasomes (caspase).[4]
The nucleotide-binding and oligomerisation domain (NOD)-like receptor (NLR) stands out among the PRRs that contribute to the establishment of the inflammasome. The human NLR family of PRRs has 22 kinds. They have a flexible N-terminal effector domain, a middle domain, and a leucine repeat-rich C-terminal region. Based on their N terminus, four NLR subfamilies are recognised: NLRA (acid activation domain), NLRB (BIR-type domain), NLRC (caspase activation and recruitment domain), and NLRP (pyrin domain).[5]
Numerous inflammasome-forming sensors respond to endogenous and microbially generated danger signals by activating inflammasomes. Inflammasomes have been investigated in connection with infectious diseases including tuberculosis and leprosy.[6] Previous research on leprosy has shown that NLRP3 is activated. Initial studies of NLRC4 utilising overexpression and co-immunoprecipitation revealed heterotypic interactions via their nucleotide binding domains with a number of NLR proteins, including NOD1, NOD2, nucleotide-binding oligomerisation domain-like receptor containing pyrin domain 3 (NLRP3), and NLR family apoptosis inhibitory protein (NAIP).[7]
Activation of the nucleotide-binding oligomerisation domain-like receptor containing caspase activation and recruitment domain (NLRC4) inflammasome seems to activate many host signalling pathways. NLRC4 was first recognised as a potential nucleotide-binding protein capable of activating caspase 1, the primary protease responsible for converting pro-IL-1β and pro-IL-18 to active interleukin 1β (IL-1β) and active interleukin 18 (IL-18) and inducing inflammatory programmed cell death, also known as pyroptosis.[8]
This study aimed to measure the concentration of NLRC4 in the blood serum of leprosy patients to identify its role in the pathogenesis of leprosy.
Materials and Methods This prospective study was carried out on 40 patients with leprosy and 30 healthy individuals of matched ages and sexes. The study was conducted at Dermatology and Venereology Department, Tanta University Hospital. The study was done after being approved by the institutional ethical committee (33216/07/19), Tanta University. Signed consent was obtained from all participants included.
Exclusion criteria were patients who had collagen disease and autoimmune disease, undergoing treatment with anti-neoplastic, corticosteroids, diabetics, pregnant, or breastfeeding.
Participants were classified into 2 different groups: Group A (patient group): included 40 patients with PB and MB leprosy. Group B (control group): included 30 healthy individuals of matched age and sex.
All patients were subjected to complete history taking including (past or family history) of similar conditions, general and dermatological examination, and laboratory investigations (CBC, Liver function test, Renal function tests, blood glucose) to exclude any systemic disease. A slit skin smear with bacillary index was done to confirm the diagnosis and determine the type of leprosy. Clinical classification of the studied patients according to WHO was based on the number of lesions[9] Paucibacillary single lesion leprosy (SLPB), PB leprosy (2–5 skin lesions), MB leprosy – six or more skin lesions, and also, all smear positive cases). Clinical classification of the studied leprosy group patients regarding disability according to WHO disability grading:[9] (Grade 0: no anaesthesia, no visible deformity or damage; Grade 1: anaesthesia present, but no visible deformity or damage; Grade 2: visible deformity or damage present). Finally, serum NLRC4 level was measured by ELISA.
Sample collection and storage: Six ml of venous blood sample was taken from each patient and delivered in a plain red-capped evacuated blood collection tube under all aseptic conditions. Samples were centrifugated, and the collected sera were kept at -20°C till use.
Principles of the Test: The serum level of NLRC4 was assayed by using a commercial kit supplied by sun red bio catalogue number SRB-T-82522, Shanghai, China. This kit implemented the sandwich ELISA method. Anti-NLRC4 antibody was pre-coated onto 96-well plates, and anti-NLRC4 antibody conjugated with horseradish peroxidase (HRP) was used as the detecting antibody. After adding the standards, test samples, and HRP-conjugated detection antibody to the wells, they were mixed, incubated, and then washed to eliminate unbound conjugates. A and B were used to test the reaction of the HRP enzyme to tetramethylbenzidine (TMB). HRP catalysed TMB to produce a blue product, which, following the addition of an acidic stop solution, became yellow. The yellow density on the plate is proportional to the amount of NLRC4 collected. The concentration of NLRC4 may then be calculated using a microplate reader to measure the optical density (OD) absorbance at 450 nm.
Assay Procedure: The components of the kit were equilibrated for 15 to 30 minutes at room temperature. On the pre-coated plate, the positions of the standard, test sample, and control (zero) wells were then recorded. 50 μl of diluted standards (1200 pg/ml, 800 pg/ml, 400 pg/ml, 200 pg/ml, and 100 pg/ml) were added to the standard wells. The control (zero) well received 100 millilitres of a normal diluent buffer (Kit Component 3). The control (zero) well did not contain any HRP-coupled antibody. Before introducing 10 μl of sample to test sample wells, 40 μl of sample diluent buffer (Kit Component 5) was added. The solution was poured into the bottom of each well without touching the sides. The dish was gently shaken to achieve a thorough blending with the plate sealer (Component 10 of Kit) and 30 minutes of incubation at 37°C. After the sealant was removed, the plate was cleaned. Each well received 50 l of an HRP-conjugated anti-NLRC4 antibody (Kit Component 6). Cover the plate with the plate sealer (Kit Component 10) and incubate at 37°C for 30 minutes. After removing the sealant, the plate must be cleaned (as in Step 5). Add 50 l of TMB chromogenic reagent A (Component 8 of the Kit) and 50 μl of TMB chromogenic reagent B to each well (Component 9 of the Kit). Shake the plate for 30 seconds on an ELISA shaker (or by hand) and incubate it at 37°C in the dark for 15 minutes. In the wells, several shades of blue are seen. Add 50 ml of stop solution (Component 7 of the Kit) to each well and well mix. The hue abruptly changes to yellow. Within 15 minutes after applying the stop solution, measure the absorbance at 450 nm using a microplate reader.
Statistical analysis
For statistical analysis, IBM's SPSS v27 (Chicago, Illinois, United States) was used. Using the Shapiro-Wilks test and histograms, the normality of the data distribution was determined. The mean and standard deviation (SD) of parametric quantitative data were explored using an unpaired student t-test. The median and interquartile range (IQR) of nonparametric quantitative data were evaluated using the Mann-Whitney test. When applicable, qualitative variables were shown as frequency and percentage and analysed using the Chi-square or Fisher's exact test. A two-tailed P value less than or equal to 0.05 was deemed statistically significant. Using Pearson's correlation coefficient for numerical variables and Spearman's rank correlation test for non-parametric variables, the correlation between two variables was found. The discriminatory function of T1 mapping for the presence of fibrosis was evaluated using a ROC curve. The level of significance was adopted at P < 0.05.
Results Demographic characteristics of the studied participants was demonstrated in [Table 1].
There was a statistically significant increase in the bacillary index in the MB group compared to the PB group (P = 0.001). Regarding the family history of the studied patients of leprosy, the increase in family history reported among MB patients in comparison to PB patients was statistically non-significant. Patients in the MB group had significantly longer durations of clinical symptoms than those in the PB group (P = 0.006). Concerning nerve involvement in the examined subgroups of leprosy, there was a statistically significant difference between PB and MB individuals. Regarding the impairment related to leprosy, there was a statistically significant difference between the PB group and the MB group among the individuals tested (P = 0.002) as shown in [Table 2].
Table 2: Comparison between the studied leprosy patients according to the bacillary index, family history, duration, nerve affection, and disability in the studied patientsNLRC4 serum level had significantly increased in leprosy patients compared to the control group (P = 0.001) [Table 3].
Table 3: Comparison between the leprosy and control groups according to the serum level of NLRC4 (ng/ml)The serum level of NLRC4 was statistically significantly different among the three studied groups (P = 0.001) [Table 4].
Table 4: Comparison between the studied leprosy subgroups and control group according to the serum level of NLRC4 (ng/ml)NLRC4 serum level showed a statistically significant increase in LL patients compared to TT patients and BL patients (p1 = 0.025), (p2 < 0.001), and (p3 = 0.001), respectively. The serum level of NLRC4 was statistically significantly different among the three studied groups (P = 0.001) [Table 5].
Table 5: Relation between the serum level of NLRC4 and the different clinical types of leprosy in studied patientsIn leprosy patients, statistically significant positive correlations were found between serum levels of NLRC4 and both the bacillary index, duration of leprosy, number of affected nerves, ages, and degree of associated disability in the studied patients (P = 0.001, 0.004, 0.001, 0.014, and 0.004 and r = 0.634, 0.340, 0.388, 0293, and 0.338), respectively [Table 6].
Table 6: Correlation between the serum level of NLRC4 (ng/ml) regarding the bacillary index and duration of leprosy, number of affected nerves, the degree of associated disability, and age (year) of the studied leprosy patientsROC curve for NLRC4 in the leprosy group (group A) was showed by using a cut-off value of 1.45 (ng/ml) for the diagnosis of leprosy. The sensitivity was 85%, specificity was 80%, and the area under the curve was 0.833 and it showed a statistically significant relation (P = 0.001) [Figure 7]a. The ROC curve for associated disability in the leprosy group (group A) was shown by using a cut-off value of 2.4 (ng/ml) for disability in leprosy patients. The sensitivity was 66.67%, the specificity was 58.33%, and the area under the curve was 0.672, and there was no statistically significant relation (P = 0.085) [Figure 7]b.
Figure 7: (a) ROC curve for serum level of NLRC4 for diagnosis of leprosy and (b) ROC curve for disability in leprosy group (group A) Discussion M.leprae causes leprosy, a chronic infectious illness. As the illness progresses, granulomatous lesions develop mostly in the skin and peripheral nerves. TT leprosy is related to the Th1 cytokine profile, while LL leprosy is associated with the Th2 cytokine profile. Borderline leprosy is characterised by variable cytokine production based on the immunological makeup of the cells. Indeterminate leprosy is the disease's initial clinical stage. Bacillus cells often engage with the immune system at this phase, hence deciding the future course of the infection.[10]
This is, as far as we are aware, the first investigation to assess the involvement of NLRC4 in the pathogenesis of leprosy.
The current study showed that all (100%) PB patients had one nerve affection, while in MB patients there were 11 (39.3%) patients without nerve affection, 6 (21.4%) patients with one nerve affection, and 11 (39.3%) patients with more than one nerve affection with a total of 29 (72.5%) of leprosy patients with nerve affection. These results are in accordance with the study conducted by Vital et al.[11] in which, 73% of the total 22 patients candidates for the study had nerve affection. Also, a study by Chaurasia et al.[12] reported that in PB leprosy, the dominant pattern of nerve involvement was that of mononeuropathy. On the contrary, other studies have reported lower values of nerve affection at the diagnosis time ranging from 9.8% in a cohort of 315 PB patients from Bangladesh[13] to 38% in North India.[14]
In PB patients, all patients (100%) had grade 1 disability. In MB patients, 12 (42.9%) patients had grade 1 disability and 3 (10.7%) patients had grade 2 disability with an overall prevalence of disability of 67.5%.
Rathod et al.[15] discovered that among 200 leprosy patients, 21.25% had a deformity of grade 1 and 6.31% had a deformity of grade 2 or worse. According to a different study by Shumet et al.,[16] the prevalence of disability among all qualifying leprosy patients was found to be 65.9% across all patient categories (40.2% grade I and 25.7% grade II).
In the current study, serum NLRC4 inflammasome levels had significantly increased in leprosy patients compared to the control group.
In agreement with our results, other studies have previously shown that the NLRP3 inflammasome (a member of the NLR family) has a role in the pathogenesis of leprosy. According to several studies, the expression of inflammasome components such as NLRP1 and NLRP3 is higher in leprosy patients.[17] However, the level of NLRC4 as an NLR component in leprosy patients has never been reported. In agreement with our results, the significant increase of NLRC4 in TT than the control group is confirmed by the presence of NLRC4 in psoriasis as both diseases are Th1 mediated.[18] This could be explained by the fact that TT is related to Th1 cytokine profile in which clones of CD4+ cells from tuberculoid patients produce high levels of IFN-γ, TNF-α, and IL-2.[1]
The significant increase of NLRC4 in LL compared to the control group is consistent with a study by Mendes et al.[19] in which immunophenotypic expression of inflammasome-associated proteins in immunopathological forms of leprosy of 99 skin lesion samples was evaluated by immunohistochemistry, and it was determined that lepromatous leprosy exhibits a strong expression of NLRP3, an NLR component similar to NLRC4.
Also, the serum level of NLRC4 showed a statistically significant increase in MB patients compared to PB patients. There was also a statistically significant positive correlation between the serum level of NLRC4 inflammasome and bacillary index in leprosy patients. This is consistent with the findings of Silva et al.,[8] who measured the amount of NLRP3 inflammasome in 43 skin lesions from leprosy patients using immunohistochemistry. LL lesions had the highest concentration of the examined marker, followed by TT lesions and ambiguous leprosy lesions.
Mendes et al.[19] reported that the high expression of NLRP3 in samples of MB forms could be attributable to the high concentration of M. leprae bacilli in these tissues and that the absence or small number of viable bacilli present in tuberculoid pole lesions was insufficient to activate the inflammasome.
In MB leprosy, the non-canonical activation of the NLRC4 inflammasome discovered in response to gram-negative bacteria such as Escherichia coli, Citrobacter rodentium, and Vibrio cholerae may occur through a similar mechanism. The outer membrane of these bacteria is mostly composed of LPS molecules, which bind and activate caspase-11, the human counterpart of caspase-4/5. It is uncertain, however, which M. leprae components may be responsible for activating this route. Inflammasomes have been established as the mechanism of IL-1β activation.[19]
NLRC4 is expressed in the human bone marrow, lymph nodes, placenta, spleen and the epidermis.[18]
The elevated level of NLRC4 in TT and LL is believed to be a protective mechanism against M. leprae. Thus, NLRC4 activation contributes to the pathogenesis of leprosy. The decreased levels of caspase 1 and IL-1β in mice with abnormal NAIP5 production, an NLR family member, show that inflammasomes are responsible for IL-1β activation. Thus, NLR protein is involved in the development of leprosy.[20]
In addition, polymorphisms in the inflammasome components NLRP1 and NLRP3 were associated with leprosy susceptibility.[21]
In addition, Stowe et al.[22] hypothesised that inflammasomes are involved in the inflammatory response caused by caspase-11 in response to bacterial infections that alter the cytoplasm of host cells, such as M. tuberculosis and M. leprae.
In the present investigation, statistically significant positive correlations were discovered between the blood concentration of NLRC4 inflammasome and the severity of nerve inflammation and disability in leprosy patients. In agreement with Jia et al.[23] findings, our data reveal that paclitaxel-induced activation of the NLRP3 inflammasome in peripheral nerves leads to neuropathic pain.
Song et al.[24] demonstrated that NLRP3 inflammasome is highly expressed in the CNS and may be triggered by many external and endogenous stimuli, including microorganisms, aggregated and misfolded proteins, and adenosine triphosphate, culminating in caspase-1 activation. The activation of caspase-1 leads to fast cell death and the processing of pro-inflammatory cytokines IL-1β and IL-18. IL-1β and IL-18 both produce neurotoxic inflammatory responses. Consequently, it is likely that NLRC4 is involved in nerve diseases.
Additionally, a statistically significant correlation between the serum concentration of the NLRC4 inflammasome and the age of the research groups was found. Inflammasome protein expression in the brains of young (3-month-old) and aged (18-month-old) mice was analysed. This study discovered that the inflammasome protein NLRC4 is more abundant in the cytosol of elderly mice's cortical lysates than in those of young mice.[25]
ROC curve for NLRC4 in the diagnosis of leprosy showed a cut-off value of 1.45 (ng/ml) and also, 85% sensitivity, 80% specificity, and 0.833 AUC. Anwar et al.[26] investigated the diagnostic accuracy of slit-skin smears as a supplementary diagnostic test for individuals with clinical suspicion of leprosy. According to the study, the diagnostic accuracy of slit skin smears was 68.75%, the sensitivity was 54.9%, the specificity was 100%, and the area under the ROC curve was 0.2%. (P value is 0.003). MB illness is only detected by skin smears in the most infectious patients and those who have clinical relapses. Slit-skin smears cannot be utilised to identify PB leprosy in clinical settings. This indicates that NLRC4 might potentially be utilised to diagnose leprosy.
The serum concentration of NLRC4 was substantially greater in leprosy patients than in controls. It was also considerably greater in MB patients compared to PB individuals. It is believed that the elevated blood level of NLRC4 in leprosy patients is a component of the defensive system against M. leprae. In addition, statistically significant positive relationships were established between the blood concentration of NLRC4 inflammasome with both nerve inflammation and disability severity. NLRC4 may thus be implicated in the aetiology of leprosy.
Limitations: It was a single-centre study with a relatively small sample size. Therefore, further studies should be done with a larger sample size using advanced technology especially research based on molecular biological methods and protein analysis techniques to better characterise the role of inflammasomes in the pathogenesis of leprosy. Additional research is also needed to evaluate the tissue expression of NLRC4 inflammasome marker of skin lesions of leprosy patients.
Conclusion: Leprosy patients had considerably greater serum levels of NLRC4 than controls. It was much greater in MB patients than in PB patients. Therefore, NLRC4 may be involved in the pathogenesis of leprosy.
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Conflicts of interest
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