Efficiency of cysticidal therapy impacted by the host’s immune response in Neurocysticercosis patients

doi:10.21203/rs.3.rs-4288121/v1

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Efficiency of cysticidal therapy impacted by the host’s immune response in Neurocysticercosis patients

https://doi.org/10.21203/rs.3.rs-4288121/v1

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published 01 Aug, 2024

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Neurocysticercosis (NCC) is a neurological condition caused by the presence of cysts from T. solium in the brain, which manifests with a range of clinical symptoms. The severity of NCC and its prognosis following Anti Helminth Drug (AHD) treatment are closely linked to peripheral and local inflammation. The study aims to analyse the efficiency of cysticidal therapy impacted by the host’s immune response in NCC patients. A total of 104 patients were enrolled in the study and blood samples were collected from 30 patients. The follow-up samples within 3 to 6 months of treatment were collected. Patients were categorised as - Responder (R) and Non-Responder (NR). Cytokines were estimated using ELISA kits in PBMCs cells. The responder patients showed significantly lower IL-10 & IL-6 levels in the supernatants at T0 as compared to T1, while in non-responder patients, IL-10 and IL-6 levels were higher at T0 as compared to T1. The IFN-γ and TNF-α levels were found to be higher in the supernatants at T0 as compared to T1 in both the responder and non-responder patients. These observations imply that these cytokines might have an impact on the efficacy of AHD treatment in individuals with NCC

Neurocysticercosis

Imaging

Antihelminth Drugs

Immune Response

Treatment

Neurocysticercosis is one of the most common neurological disorders and a public threat in many portions of the world (Butala et al., 2021). The condition arises due to the presence of cysts from Taenia solium in the brain and exhibits various clinical manifestations. The genetic background of the host and the inflammatory reaction of the host play a role in determining the severity of the disease (Ta and Blond 2022). Inflammatory responses in Neurocysticercosis vary depending on the location of the cysts. Once established within the Central Nervous System (CNS), the cysticercus triggers an immunomodulatory reaction, dampening the inflammatory response and facilitating the parasite's survival (Prodjinotho et al., 2020). However, when degeneration and death of the cysticercus occur, this immunomodulation is disturbed, provoking an inflammatory reaction linked to severe NCC symptoms like seizures and encephalitis.

Therefore, it is crucial to manage NCC treatment carefully to prevent adverse inflammatory outcomes in the CNS (WHO, 2021). Despite the widespread impact of NCC globally, many aspects of the disease remain uncertain. Many clinicians do not prescribe Anti Helminth Drugs (AHD) without brain imaging because of the great risk linked with the inflammatory response which varies with the location and number of the viable cysts present. The severity of the disease and its prognosis are closely correlated with both the intensity of the host's inflammatory response against the parasite and the localization of cysticerci (Fleury et al., 2004). Due to the significance of neuroinflammation in the development of NCC, extensive efforts have been made to understand and characterize the immune-inflammatory response in NCC and its role in combating the parasite

An AHD seems to be effective and in fact, a single cycle of treatment can make cysticerci calcified or disappear. But in the case of extra parenchymal NCC, when cysticerci present in the subarachnoid space of the base, the symptoms shown are diverse such as hydrocephalus, vasculitis, and arachnoiditis. In such cases, AHD treatment demonstrates limited effectiveness, with nearly one-third of patients requiring more than two cycles of treatment (Fleury et al., 2011). The reason behind this poor response to AHD is yet to be elucidated. As the simultaneous administration of the corticosteroid which helps to reduce severe inflammation might be involved. Corticosteroids, being immunosuppressive, reduce the host's specific immunity against the parasites. This specific immunity, in conjunction with AHD, is necessary to eradicate the parasite (Guilpain et al., 2012). All these observations drive us to investigate the relevance of differential responsiveness to cysticidal drug treatment accompanying the immune inflammatory profile.

Variations are found in the immunological profile of various forms of NCC such as in the case of Parenchymal NCC in which the TH2 profile is dominant while in Extraparenchymal NCC depressed lymphocyte proliferative response is observed. The severity of Neurocysticercosis and its prognosis following AHD treatment are closely linked to peripheral and local inflammation. So, the objective of this study is to analyze the immune profile of patients before and after treatment, to assess the response of AHD concerning host immune response. This observation may imply a potential impact of cytokines on the efficacy of AHD treatment, as indicated by radiological findings.

It implies a possible role of the host's immune system in determining the efficacy of AHD treatment.

Patients Recruitment

The inclusion criteria for the patients comprised those who were diagnosed radiologically through imaging methods (CT&MRI), showed the presence of a viable parasite, received AHD treatment, and underwent clinical follow-up for 3–6 months after initiating the first cycle of treatment. Patients with calcified cysts or those who had already started AHD treatment were excluded from the study. Based on the imaging method and treatment response, the patients were categorized as Responder (R) or Non-Responder (NR). Responders were defined as patients with a reduction exceeding 50% in parasitic burden, while non-responders were identified with a reduction of less than 50%.

Preparation of ES Antigen of Taenia solium

The viable cysts extracted from infected pig muscle with transparent membrane and visible scolex were observed under the microscope. These cysts were washed for a maximum of four hours in a PBS solution containing antibiotics. In vitro, cultivation of T. solium cysticerci was done in RPMI-1640 media. After every 24 hrs medium was replaced and the medium removed was collected. The media was concentrated using a protein concentrator. RIPA lysis buffer was added to the concentrated protein. After adding the RIPA buffer, the lysate was centrifuged for 30 min at 15000g and 4°C to remove non-lysed fragments. Then the lysate was sonicated for 1 min and the aliquots were stored at -20⁰C for further use. Protein Concentration was checked using Bradford Assay.

Peripheral Blood Mononuclear Cells (PBMCs) isolation and culture: 5 mL of blood was drawn from the peripheral veins and collected in an EDTA tube. Blood was 1:2 diluted with RPMI medium. Ficoll-Hypaque was layered at the top and centrifuged. The cell viability was checked by Trphan blue staining. Cells present in four squares of the Neubauer chamber were counted. The viable cells were seen as bright cells and nonviable as blue stained. The concentration of viable, and nonviable cells and viability was calculated (Fig. 2). PBMCs in vitro culture was performed. For that isolated cells were suspended in RPMI medium in 6 well flat-bottom culture plates (Fig. 1) for 72–120 hours. The RPMI medium was prepared using a powdered RPMI mixture (Sigma). Culture media was supplemented with 10% human AB serum, 1% non-essential amino acids, 1% pyruvate, 2 mM l-glutamine, 100 ng/mL streptomycin, and 100 U/mL penicillin. The cells were stimulated by 30–35 µg/mL per well for every 2.5 million cells of Taenia solium ES Antigen (TsAg). The culture well plates were maintained at 37°C in a 5% CO2 and humidity for incubation.

Cytokine Determination

Cultured PBMCs stimulated in vitro with ES TsAg were centrifuged and the supernatant was collected in a 15ml tube. The supernatant was centrifuged for 10 minutes at 1000×g to remove all the particulates and aggregates. Cytokines IFNɣ, TNFα, IL-10, and IL-6 were estimated using Diaclone Human ELISA Kits which works on the solid phase sandwich ELISA principle. Absorbance was read by an ELISA reader (Biotek µquant) at 450nm as the primary wavelength and 620nm as the reference wavelength. A linear standard curve was plotted, in which the absorbance of every standard was at the vertical axis versus a standard concentration of corresponding cytokine (IFNɣ, TNFα, IL-10, IL-6) on the horizontal axis. For each set of microwell strip assays standard curve was plotted individually.

ELISA data were presented as the median with a 95% confidence interval and analysed using the two-tailed Student’s t-test with a significance threshold of p < 0.05.

A study was conducted with a total of 104 patients initially enrolled, of which only 30 met the inclusion criteria. Among these 30 patients, 26 attended the next follow-up. MRI/CT scan of patients was done within the period of 3 to 6 months of the first follow-up. 21 patients were categorized into the responder category and 5 patients were categorized into the nonresponder category. In the responder patients, 15 patients had viable cysts which became calcified while in 6 patients the viable cyst disappeared within 6 months of treatment. In the non-responder patients, out of 5 patients, 3 patients had intact cysts while in 2 patients the cyst size was decreased after 6 months of treatment.

Comparison of IL-10 and IL-6 levels between responder and non-responder patients (pre-treated T0 vs. post-treated T1 groups (Graph 1&2): Responder patients exhibited significantly lower levels of IL-10 and IL-6 in the supernatant at T0 compared to T1. Conversely, in non-responder patients, IL-10 and IL-6 levels were higher at T0 compared to T1 (Graph 6.15). The mean value of IL-10 for responders at T0 stages i.e., pretreatment is 38.6 (93.4-27.6), and at T1 i.e., post treatment is 227.0 (1087.9-85.2). In non-responders, the mean value at T0 is 51.8(75.2-34.4) while at T1 is 36.0 (37.3-34.4). The mean value of IL-6 for responders at T0 is 30.3 (77-13.8) and at T1 is 108.4 (544-25.4). In non-responders, the mean value at T0 is 28.2 (35.4-19.3) while at T1 is 22.8 (27.8-17.9) (Table 1)

Comparison of IFN -γ and TNF- α levels between responder and non-responder patients (Pre-treated T0 vs. post-treated T1 groups) (Graph 3&4)

The IFN -γ and TNF- α levels were found higher in the supernatant at T0 as compared to T1 in both responder and non-responder patients (Graph 6.17.). The mean value of IFN -γ for responders at T0 is 70.8 (507.1–29.6) and at T1 is 39.8(197.2–22). In non-responders the mean value at T0 is 29.6 (37.8–23.8) while at T1 is 25.5 (28.5–22.2) (Table 6.7). The mean value of TNF- α for responders at T0 is 109.8(785.4–30.3) and at T1 is 35.7(208.9–12.8). In non-responders the mean value at T0 is 51.3 (114.8–23.9) while at T1 is 31.4 (66.6–11.4) (Table 1).

Table 1

Comparison of cytokine levels between responders and non- responders at T0 and T1 in NCC patients
Variables	Mean	Minimum	Maximum	Lower 95% CI of mean	Upper 95% CI of mean	Univarirate Sig.	Sig.
IFN-ɣ Responder T0	70.86	29.68	507.1	8.629	133.1	< 0.0001***	0.0008**
IFN-ɣ Responder T1	39.79	22.05	197.2	17.02	62.56	< 0.0001***	0.0008**
IFN-ɣ Non-Responder T0	29.63	23.81	37.75	24.82	34.44	0.0133	0.0729
IFN-ɣ Non-Responder T1	25.5	22.19	28.51	23.28	27.72	0.0133	0.0729
IL-10 Responder T0	38.58	27.65	93.42	30.38	46.78	< 0.0001***	< 0.0001***
IL-10 Responder T1	227	85.2	1088	68.4	385.6	< 0.0001***	< 0.0001***
IL-10 Non-Responder T0	51.8	34.4	75.22	35.47	68.14	0.3207	0.0323*
IL-10 Non-Responder T1	35.97	34.4	37.34	34.76	37.18	0.3207	0.0323*
IL-6 Responder T0	30.34	13.82	76.95	19.44	41.25	0.0002	0.0002***
IL-6 Responder T1	108.4	25.42	544	28.28	188.5	0.0002	0.0002***
IL-6 Non-Responder T0	28.24	19.26	35.41	22.16	34.31	0.0023	0.085
IL-6 Non-Responder T1	22.8	17.93	27.77	18.73	26.87	0.0023	0.085
TNF-α Responder T0	109.8	30.26	785.4	10.88	208.8	< 0.0001***	< 0.0001***
TNF-α Responder T0	35.72	12.84	208.9	10.21	61.24	< 0.0001***	< 0.0001***
TNF-α Non-Responder T0	51.32	23.87	114.8	17.15	85.48	0.0133	0.132
TNF-α Non-Responder T1	31.37	11.38	66.56	11.05	51.69	0.0133	0.132
All statistical tests used in this study were two-tailed, with a significance level of p < 0.05 (represented as p < 0.05, p < 0.01, ***p < 0.001).

The study examined the cytokine profile of both responder and non-responder patients with NCC. The cytokines IL-6, IL-10, TNF- α, and IFN -γ were studied using Sandwich ELISA kits IL-6, a proinflammatory cytokine, is essential in mediating immune responses and inflammation. The study conducted by Chavarría et al., 2005 showed elevated levels of IL-6 in the CSF of NCC patients. In the present study, responder patients had lower IL-6 levels before treatment (T0) compared to after treatment (T1). After treatment, the IL-6 levels increased in the responder group. The increase in levels of IL-6 after treatment in the responding group could be indicative of an immune response or an inflammatory reaction triggered by the treatment, potentially contributing to the positive response observed. In non-responder patients, IL-6 levels were high before treatment (T0) and decreased after treatment (T1) but the decrease in levels was not significant. This may suggest that the treatment had some impact on the immune response, even if it did not result in a favorable treatment outcome in terms of response to therapy. IL-10 functions as an anti-inflammatory cytokine, regulating the immune response. IL-10 is associated with immunoregulatory processes and may have both protective and detrimental effects on NCC. The study conducted by Harrisson et al., 2021 suggested high levels of IL10 levels in the cured subarachnoid Neurocysticercosis patients. In the present study, responder patients had lower IL-10 levels before treatment (T0) compared to after treatment (T1). After treatment, the IL-10 levels increased in the responder group. This indicates that the treatment resulted in elevated IL-10 levels among the responsive patients. The increase in levels of IL-10 after treatment in the responder group could be indicative of a regulatory response aimed at dampening inflammation and promoting tissue repair. In contrast, non-responder patients had higher IL-10 levels before treatment (T0) and experienced a decrease in IL-10 levels after treatment (T1). The decrease in IL-10 levels in the non-responder group after treatment could reflect a reduction in the regulatory response or modulation of the immune response that was not sufficient to achieve a positive treatment outcome. Tumor Necrosis Factor-alpha (TNF-α) and Interferon-gamma (IFN-γ) are pro-inflammatory cytokines known for their roles in the formation and maintenance of granulomas, which are characteristic features of NCC. In our study, the levels of TNF-α and IFN-γ were found to be higher in the supernatant at T0 compared to T1 in both responder and non-responder patients. This implies that the treatment resulted in a decrease in cytokine levels across both groups, suggesting a reduction in inflammation and immune response among both responder and non-responder patients. These findings regarding cytokine levels in responder and non-responder groups align with those of a study conducted in Mexico by Cárdenas et al. in 2014, which examined cytokine levels in the CSF and PBMC supernatant of NCC patients.

The study aimed to investigate the immune-inflammatory characteristics associated with the efficacy or lack of efficacy of AHD treatment by assessing cytokine levels in NCC patients categorized as Responders (R) or Non-responders (NR). The results of our study emphasize the potential significance of certain cytokines in the successful outcome of AHD treatment. These findings suggest that these cytokines might have an impact on influencing the effectiveness of AHD treatment in NCC patients.

Competing interests:

The authors have no relevant financial or non-financial interests to disclose.

Ethics Approval and Consent to Participate:

The Ethics Committee of PGIMER, Chandigarh, approved the study vide letter no. NK/6722/PhD/478.

Consent for Publication:

Informed consent was obtained from all individual participants included in the study.

Conflict of Interest:

I declare that none of the authors have any conflicts of interest to disclose pertaining to this manuscript

Funding:

Funding Support was provided by Indian Council of Medical Research (ICMR), New Delhi.

Author Contribution

Yashvi Mehta: Conceptualization; Data curation, Project administration, original draft writing Upninder Kaur: Formal analysis, Review, and editing, ResourcesRitu Shree: Visualization, Review and Editing Manish Modi: Formal analysis, SupervisionParamjeet Singh: Conceptuaization and Supervision Vivek Lal: Investigation, SupervisionRakesh Sehgal: Investigation, Supervision and Validation

Data Availability:

The data supporting the findings can be obtained upon request from the corresponding author

Butala C, Brook TM, Majekodunmi AO, Welburn SC (2021) Neurocysticercosis: current perspectives on diagnosis and management. Front Vet Sci 8:615703
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Prodjinotho UF, Lema J, Lacorcia M et al (2020) Host immune responses during Taenia solium Neurocysticercosis infection and treatment. PLoS Negl Trop Dis 14(4):e0008005
World Health Organization (2022) WHO Taenia solium endemicity map–2022 update–Carte OMS d’endémicité de Taenia solium–Mise à jour 2022. Wkly Epidemiol Rec 97(17):169–172
Fleury A, Dessein A, Preux PM, Dumas M, Tapia G, Larralde C, Sciutto E (2004) Symptomatic human neurocysticercosis: age, sex and exposure factors relating with disease heterogeneity. J Neurol 251:830–837
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Harrison S, Thumm L, Nash TE, Nutman TB, O’Connell EM (2021) The local inflammatory profile and predictors of treatment success in subarachnoid Neurocysticercosis. Clin Infect Dis 72(9):e326–e333
Cárdenas G, Fragoso G, Rosetti M, Uribe-Figueroa L, Rangel-Escareño C, Saenz B, Hernández M, Sciutto E, Fleury A (2014) Neurocysticercosis: the effectiveness of the cysticidal treatment could be influenced by the host immunity. Med Microbiol Immunol 203:373–381

Graphs 1 to 4 are available in the Supplementary Files section.

No competing interests reported.

GR1.png
Graph 1. Comparison of IL10 levels between Responder and Non Responder patient
GR2.png
Graph 2. Comparison of IL-6 levels between Responder and Non-Responder patients
GR3.png
Graph 3. Comparison of IFN-ɣ levels between Responder and Non Responder patients
GR4.png
Graph 4. Comparison of TNF-α levels between Responder and Non-Responder patients

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Efficiency of cysticidal therapy impacted by the host’s immune response in Neurocysticercosis patients

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