Strongyloides stercoralis coinfection impairs immune control of Mycobacterium tuberculosis in TB infected individuals

Background: Helminth co-infection are common in tuberculosis (TB) endemic regions and alter host immunity. However, their impact on immune responses in TB infection (TBI) remains incompletely defined.

Methods: We analyzed QuantiFERON (QFT) supernatants and peripheral blood mononuclear cells (PBMCs) from TBI+Hel+ (S. stercoralis (Ss) infection) (n=15) and TBI+Hel− (n=23) individuals. Cytokine levels were assessed at baseline and following TB antigenic stimulation (TBAg1 and TBAg2), as well as mitogenic stimulation. PBMCs were stimulated with Mycobacterium tuberculosis (M. tuberculosis) H37Rv, and antimycobacterial immunity was assessed using an in vitro mycobacterial growth inhibition assay (MGIA) and multiplex cytokine assays.

Results: We measured Type 1 (IFN-γ, TNF-α, IL-2), Type 17 (IL-17, IL-22), and proinflammatory cytokines (IL-1α, IL-1β) in QuantiFERON (QFT) supernatants from TBI individuals with (TBI+Hel+) and without (TBI+Hel−) infection. TBI+Hel+ individuals exhibited significantly reduced mycobacterial growth inhibition compared with TBI+Hel− individuals, reflected by higher normalized mycobacterial growth (Δ log10 CFU). Cytokine analysis showed suppression of Th1/Th17 (IFN-γ, IL-2, TNF-α, IL-17, GM-CSF) and proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-12p70, IL-18) in TBI+Hel+ individuals, alongside increased Th2/regulatory cytokines (IL-4, IL-5, IL-10, IL-13).

Conclusions: Ss co-infection impairs host antimycobacterial control by skewing TBI immune responses toward Th2/regulatory pathways at the expense of protective Th1/Th17 mechanisms. Our data demonstrate that the importance of considering Ss co-infection into account when developing TB control and vaccination strategies in endemic areas.

1 Introduction

Helminth infections affect approximately 1.5 billion people worldwide, predominantly in tropical regions, and typically induce a T-helper 2 (Th2) immune response characterized by IL-4, IL-5, and IL-13 (1). TB infection (TBI), the asymptomatic phase of Mycobacterium tuberculosis (M. tuberculosis) infection, presents a significant public health challenge due to its potential for reactivation, especially in immunocompromised individuals (2). Approximately 25% of the global population has TBI, and an estimated 5% progress to active TB within two years of infection (https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/tb-reports/global-tuberculosis-report-20243). A Th1-mediated immune response, involving cytokines such as IFN-γ and TNF-α, is critical for controlling Mtb (2).

Helminths may shift immune responses to M. tuberculosis, favoring Th2/regulatory pathways and potentially impairing Th1-mediated control (4, 5). Conversely, some studies suggest helminths might reduce inflammation and tissue damage during active TB, potentially offering protective effects (5, 6). However, the exact mechanisms through which helminths impact TB progression remain unclear.

The Mycobacteria Growth Inhibition Assay (MGIA) is a key tool for evaluating immune responses to M.tb, providing insights into the Th1/Th2 balance and how helminth infections modify this response (7). Cytokine profiles from MGIA assays show that M.tb typically induces a Th1 response, while helminths promote a Th2 environment, potentially hindering the immune mechanisms necessary for effective M.tb control. This shift could lead to increased susceptibility to TB reactivation (2, 4, 8).

Understanding how helminths modulate immune responses in TBI is critical for predicting the outcome of co-infection. Helminth-induced regulatory cytokines, such as IL-10, may suppress Th1 responses, impairing the host’s ability to contain Mtb (9). However, some studies also suggest that helminths could reduce immune hyperactivation, potentially minimizing tissue damage in TB (10). Recent studies further reveals that helminth co-infection modulates chemokine and cytokine responses in TBI, with implications for disease progression (5, 11).

Our study aims to investigate cytokine profiles from MGIA assays in individuals with TBI and Ss co-infection. In our study population, all helminth infections identified were caused by Strongyloides stercoralis; therefore, results refer specifically to S. stercoralis co-infection. The goal is to explore how Ss induced immune modulation affects the Th1/Th2 balance and M.tb growth inhibition, providing insights that could enhance TB management strategies in endemic regions.

2 Materials and methods

2.1 Ethical statement

Ethical approval was obtained from the Institutional Review Boards of the National Institute of Allergy and Infectious Diseases (NIAID), USA, and the National Institute for Research in Tuberculosis (NIRT), India (NCT04813328, NCT04526613, NIRT-IEC 2020–033 and NIRT-IEC 2020 005). Written informed consent was obtained from all study participants.

2.2 Study population

We enrolled 38 individuals with TB infection (TBI) from Tiruvallur and Kanchipuram Districts, Tamil Nadu, South India, between September 2022 and January 2023: 15 with Ss co-infection (TBI+Hel+) and 23 without Ss co-infection (TBI+Hel−). Participants were aged 22–63 years. These 38 individuals were prospectively enrolled specifically for this study and were not selected from a larger pre-existing cohort. TBI diagnosis was based on positive QuantiFERON-TB Gold Plus (QFT-Plus) results (QIAGEN), absence of pulmonary symptoms, and normal chest radiographs. None of the participants were known contacts of active TB cases. Pregnant or lactating women were excluded from the study. All participants were HIV-negative and anti-TB treatment–naïve. TBI was identified for study enrolment; TBI-positive individuals were referred to TB clinics per public health protocols.

2.3 QuantiFERON-TB gold in-tube assay

QFT-Plus testing followed manufacturer guidelines. Heparinized whole blood was incubated at 37°C for 18 hours in TB1, TB2, mitogen, and Nil tubes. In the QFT-Plus assay (Qiagen), the TB1 tube contains long ESAT-6 and CFP-10 peptides that predominantly stimulate CD4⁺ T cell responses, whereas the TB2 tube contains additional short ESAT-6 and CFP-10 peptides designed to activate both CD4⁺ and CD8⁺ T-cell subsets. Tubes were centrifuged at 3,000 g for 15 minutes, and supernatants were collected for IFN-γ measurement (IU/mL). Remaining supernatants were stored at −80°C.

2.4 Parasitological examination and anthelmintic treatment

Strongyloides stercoralis (Ss) infection was diagnosed by IgG antibodies to the recombinant NIE antigen (12, 13). A single stool sample was examined by Kato-Katz; stool microscopy was negative for other intestinal helminths. Ss infection was further confirmed by nutrient agar plate culture (14). Filarial infection was excluded by negative circulating filarial antigen tests. S. stercoralis was the only helminth species detected in this study population. All Ss-positive participants received ivermectin (12 mg, single dose) and albendazole (400 mg, single dose). Follow-up blood draws and repeat parasitological examinations were performed six months later to confirm clearance.

2.5 Mycobacterial growth inhibition assay

M.tb H37Rv was cultured in a Middlebrook 7H9 liquid medium (Sigma-Aldrich). The mycobacterial inoculation volume for each batch was titrated to achieve a time to positivity (TTP) of 7 days in MGITs in a BACTEC MGIT 960 machine. The inoculum was titrated to achieve a 7-day time to positivity (TTP) in MGITs; inocula ranged between approximately 10^2–10^3 CFU/mL.

2.6 Isolation of PBMCs and mycobacterial growth inhibition assay

Human peripheral blood mononuclear cells were isolated from peripheral blood and resuspended at an approximate concentration of 2–3 × 10⁶ cells per mL of RPMI (containing 10% fetal bovine serum and 2 mM l-glutamine) and benzonase was added to a final concentration of 25 U/mL. Cells were rested at 37°C for 2 hours with 5% CO₂. Six hundred microliters of RPMI (containing 2 mM L-glutamine and 25 mM HEPES) seeded with 1×10⁶ PBMC and M.tb H37Rv was added to duplicate 2-mL screw-cap tubes. The co-cultures were incubated on a 360° rotator at 37 °C for 96 hours, after which tubes were micro-centrifuged at 15,300 g for 10 minutes, and the supernatant was carefully removed by pipetting. Cells were lysed with the addition of 500 µL sterile water and the tubes pulse-vortexed at 0, 5 and 10 minutes. Five hundred microliters of lysate were then transferred directly to a BACTEC MGIT tube supplemented with PANTA antibiotics (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) and OADC enrichment broth. On day 0, duplicate direct-to-MGIT inoculum control tubes were prepared with the same M. tuberculosis input as samples. In the end, MGIT tubes were placed on the BACTEC 960 machine and incubated at 37 °C until the detection of positivity by fluorescence. TTP was converted to log10 CFU using standard curves generated by inoculating MGIT tubes with 10-fold mycobacterial dilutions and plating aliquots on 7H11 agar. Linear regression (GraphPad Prism v10) provided equations for TTP-to-CFU conversion. Normalized mycobacterial growth was defined as log10 CFU (sample) − log10 CFU (growth control). The difference between the medians of respective groups is described as Δ X log and was calculated by subtracting the median of the test group from the median of the control group.

2.7 Luminex assays for cytokines

Cytokine levels in culture supernatants were measured using a Human Magnetic Luminex Assay (R&D Systems) on the Bio-Rad MAGPIX platform (xPONENT 4.2; Bio-Plex Manager 6.1). Analytes included Interferon gamma (IFNγ), interleukin-2 (IL-2), Tumor Necrosis Factor alpha (TNFα), IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-6, IL-12p70, IL-18, IL-4, IL-5, IL-10, and IL-13. The lowest detection limits for cytokines were as follows: GM-CSF, 18.4 pg/mL; IFN-γ, 5.7 pg/mL; IL-1α/IL-1F1, 10.6 pg/mL; IL-1β/IL-1F2, 3.5 pg/mL; IL-2, 3.6 pg/mL; IL-4, 1.1 pg/mL; IL-5, 6.2 pg/mL; IL-6, 9.0 pg/mL; IL-10, 32.2 pg/mL; IL-12p70, 18.5 pg/mL; IL-13, 31.8 pg/mL; IL-17/IL-17A, 9 pg/mL; IL-18/IL-1F4, 2.5 pg/mL; and TNF-α, 12.4 pg/mL.

2.8 Statistical analysis

Data were summarized as medians with interquartile ranges (IQR). Between-group comparisons used the Mann–Whitney U test with Holm–Bonferroni correction for multiple comparisons. Correlations were assessed using Spearman’s rank correlation. Ss-specific IgG (OD values) and all cytokines measured in QFT or PBMC supernatants were entered as independent variables in a multivariate linear regression model to assess their combined association with MGIA (Δ log10 CFU). Analyses were performed in GraphPad Prism v10.0; significance was set at p ≤ 0.05.

3 Results

3.1 Study population characteristics

The demographic, hematological, and biochemical characteristics of the study participants are summarized in Table 1. There were no statistically significant differences in age, gender, or body mass index (BMI) between the Tuberculosis infected (TBI) individuals with helminth infection (TBI+ Hel+) and those without helminth infection (TBI+). As only Strongyloides stercoralis infection was identified among helminth-positive participants, the comparisons presented here specifically reflect the impact of S. stercoralis co-infection on immune responses in TBI. However, notable differences were observed in certain hematological parameters. TBI+ Hel+ individuals exhibited significantly decreased levels of hemoglobin (TBI+ Hel+ 11.25 Vs TBI + 13.10 g/dl, p=0.022), Red Blood Cells (RBC) (TBI+ Hel+ 4.75 Vs TBI + 4.05 (10³/ml), p=0.034) and lymphocytes compared to TBI+ individuals. In contrast, eosinophil levels (TBI+ Hel+ 0.75 Vs TBI + 0.44 (10³/ml), p=0.028 were significantly increased in TBI+ Hel+ individuals compared to TBI+ individuals.

Table 1

Table 1. Demographic and hematological parameters of the study population.

3.2 Decreased Th1, Th17, and proinflammatory cytokine responses in Ss co-infected TBI individuals

To determine the influence of coexistent Ss infection on type 1, type 17, and proinflammatory cytokines in TBI, we measured the QFT supernatants levels of IFN-γ, TNF-α, IL-2, IL-17, IL-22, IL-1α and IL-1β in TBI+Hel+ and TBI+Hel- individuals. As shown in Figure 1, TBI+Hel+ co-infected individuals exhibited significantly decreased levels of Type 1, median (IFN-γ: TBI+Hel+, 63 pg/mL Vs TBI+, 268.1 pg/mL, p=0.0126, IL-2, TBI+Hel+, 195 pg/mL Vs TBI+, 265 pg/mL, p=0.0267; TNF-α: TBI+Hel+, 25.06 pg/mL Vs TBI+, 47.50 pg/mL, p=0.0177; Type 17 (IL-17, TBI+Hel+, 10 pg/mL Vs TBI+, 16 pg/mL, p=0.0035; IL-22, TBI+Hel+, 15.88 pg/mL Vs TBI+, 23.56 pg/mL, p=0.0306 and proinflammatory cytokines, IL1α, TBI+Hel+, 3.45 pg/mL Vs TBI+, 4.07 pg/mL, p=0.0233 and IL1β, TBI+Hel+, 15.74 pg/mL Vs TBI+, 17.42 pg/mL, p=0.0365 at baseline and upon TBAg1 stimulation, median (IFN-γ: TBI+Hel+, 267.5 pg/mL Vs TBI+, 415.1 pg/mL, p=0.0312, IL-2, TBI+Hel+, 175 pg/mL Vs TBI+, 352.9 pg/mL, p=0.0181; TNF-α: TBI+Hel+, 27.38 pg/mL Vs TBI+, 61.11 pg/mL, p=0.0395; and IL-22, TBI+Hel+, 98 pg/mL Vs TBI+, 124 pg/mL, p=0.0240; upon TBAg2 antigenic stimulation median (IL-2, TBI+Hel+, 230 pg/mL Vs TBI+, 381.2 pg/mL, p=0.0086; TNF-α: TBI+Hel+, 40.99 pg/mL Vs TBI+, 68 pg/mL, p=0.0217; Type 17 (IL-22, TBI+Hel+, 28.20 pg/mL Vs TBI+, 78.20 pg/mL, p=0.0084; proinflammatory cytokines, IL1α, TBI+Hel+, 4 pg/mL Vs TBI+, 5.5 pg/mL, p=0.0145 and IL1β, TBI+Hel+, 15.48 pg/mL Vs TBI+, 19.80 pg/mL, p=0.0220 in comparison with TBI individuals alone. In contrast, the upon mitogenic stimulation there was no significant difference between the two groups. Thus, coexistent Ss infection is associated with diminished systemic levels of type 1 type 17 cytokines and proinflammatory cytokines in TBI alone individuals.

Figure 1

Figure 1. Decreased Th1, Th17, and proinflammatory cytokine responses in Ss co-infected TBI individuals.Helminth infection is associated with diminished systemic levels of type 1 and type 17 cytokines TBI. Panels (A–D) show QFT supernatant cytokine levels at baseline (Nil), TB antigen 1 (TB1), TB antigen 2 (TB2), and mitogen stimulation in TBI+Hel+ (n=15) and TBI+Hel− (n=23) individuals. Data are displayed as medians with IQR; p values from Mann–Whitney U with Holm–Bonferroni correction.

3.3 Decreased mycobacterial growth inhibition in Ss infected LTBI individuals

We assessed the in vitro growth inhibition of Mycobacterium tuberculosis H37Rv in peripheral blood mononuclear cells (PBMCs) from individuals with latent tuberculosis infection (TBI) with and without Ss co-infection. As shown in Figure 2, TBI+Hel+ individuals exhibited significantly higher normalized mycobacterial growth (Δ log10 CFU 3.8) than TBI+Hel− individuals (Δ log10 CFU 2.8; p=0.0127), indicating reduced capacity to inhibit M. tuberculosis growth in the Ss co-infected group.

Figure 2

Figure 2. Mycobacterial growth inhibition assay in tuberculosis infected individuals with Ss infection. The figure displays MGIA normalized mycobacterial growth (Δ log10 CFU) in TBI+Hel+ and TBI+Hel− individuals. Each point represents one participant; bars show medians. Mann–Whitney U test with Holm–Bonferroni correction was used for all pairwise comparisons.

3.4 Ss co-infection suppresses both adaptive and innate cytokine responses to Mycobacterium tuberculosis

We evaluated cytokine responses following stimulation of PBMCs with Mycobacterium tuberculosis H37Rv to assess the impact of Ss co-infection in individuals with tuberculosis infection (TBI). As shown in Figure 3, TBI-helminth co-infected individuals exhibited significantly diminished secretion of Th1 cytokines, median concentrations were IFN-γ (35 pg/mL Vs 26.51 pg/mL), IL-2 (7.6 pg/mL Vs 6.8 pg/mL), and TNF-α (39.1 pg/mL Vs 33.8 pg/mL), compared to TBI+ individuals alone. Furthermore, median concentrations were IL-17 (139.2 pg/mL Vs 119.0 pg/mL) and GM-CSF (21.08 pg/mL Vs 18.2 pg/mL) —key cytokines involved in Th17-mediated immunity and granuloma integrity—were also significantly reduced in the helminth-infected group.

Figure 3

Figure 3. Cytokine secretion profile in tuberculosis infected individuals with Ss infection. The figure illustrates the Type 1 and pro-inflammatory cytokine secretion profile in response to M. tuberculosis H37Rv in TBI+Hel+ and TBI+Hel- individuals. Cytokines including Interferon gamma (IFNγ), Interleukin-2 (IL-2), Tumor necrosis factor alpha (TNF-α), Interleukin-17 (IL-17), Granulocyte macrophage colony-stimulating factor (GM-CSF), Interleukin-1 alpha (IL-1α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Interleukin-12 (IL-12p70), Interleukin-18 (IL-18), were measured in the culture supernatant. Each data point represents an individual subject, with the bar indicating the geometric mean (GM) cytokine level. Statistical analysis was performed using the Mann–Whitney U-test with Holm–Bonferroni correction was used for all pairwise comparisons.

In addition to the suppression of adaptive immune responses, innate inflammatory cytokine production was also markedly impaired. Notably, levels of median concentration were IL-1α (179.7 pg/mL Vs 144.9 pg/mL) and IL-6 (201.2 pg/mL Vs 132.3 pg/mL), were significantly decreased in LTBI-helminth co-infected individuals following M. tuberculosis stimulation. Whereas IL-1β (16.8 pg/mL Vs 13.88 pg/mL), IL-12p70 (49.2 pg/mL Vs 43.8 pg/mL) and IL-18 (5.7 pg/mL Vs 5.3 pg/mL) levels were decreased in TBI- Ss co-infected individuals but did not show any significant difference when compared with TBI+ individuals alone. These cytokines are critical for early-phase immune activation and effective initiation of host defense mechanisms against TB. Together, these results indicate that Ss co-infection leads to broad immune suppression encompassing both innate and adaptive arms of the immune response to M. tuberculosis.

3.5 Enhanced type 2 and regulatory cytokine responses

Next, we evaluated the Th2 and regulatory cytokines following stimulation of PBMCs with Mycobacterium tuberculosis H37Rv to assess the impact of Ss co-infection in individuals with tuberculosis infection (TBI). In contrast, Ss infected individuals exhibited elevated levels of type 2 and regulatory cytokines, median concentrations were IL-4 (5.1 pg/mL Vs 7.1 pg/mL), IL-5 (21.2 pg/mL Vs 26.4 pg/mL), IL-10 (142.9 pg/mL Vs 204.3 pg/mL), and IL-13 (47.9 pg/mL Vs 50.6 pg/mL), following M. tuberculosis stimulation (Figure 4). These findings are consistent with a Ss induced immunological milieu that favors Th2 and regulatory responses, potentially at the cost of effective antimycobacterial immunity.

Figure 4

Figure 4. Type 2 cytokine secretion profile in tuberculosis infected individuals with Ss infection. The figure illustrates the Type 2 cytokine secretion profile in response to M. tuberculosis H37Rv in TBI+Hel+ and TBI+Hel- individuals. Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-10 (IL-10), and Interleukin-13 (IL-13) were measured in the culture supernatant. Each data point represents an individual subject, with the bar indicating the geometric mean (GM) cytokine level. Statistical analysis was performed using the Mann–Whitney U-test with Holm–Bonferroni correction was used for all pairwise comparisons.

3.6 Principal component analysis of cytokine and MGIA responses

Principal component analysis (PCA) was performed to identify patterns of immune variation associated with helminth coinfection in TBI individuals (Figure 5). The first two components (PC1 and PC2) accounted for approximately 44.6% of the total variance (PC1: 28.6%, PC2: 16%), capturing the major immunological differences between the study groups. The PCA biplot showed distinct clustering between TBI and TBI⁺+Hel⁺ group, indicating that Ss infection significantly altered the cytokine response network. Cytokines such as IL-4, IL-5, IL-10, and IL-13 loaded strongly on the positive axis of PC1, aligning with MGIA, and reflected a Th2/regulatory-dominant profile in helminth-infected individuals. In contrast, IL-17, IFN-γ, IL-2, TNF-α, IL-1α, and IL-1β clustered negatively along PC1, representing Th1/Th17-associated inflammation characteristic of helminth-negative TBI cases. This segregation highlights that helminth infection promotes a systemic shift from Th1/Th17 to Th2/regulatory dominance, consistent with reduced mycobacterial control.

Figure 5

Figure 5. Principal component analysis (PCA) of cytokine and MGIA responses in tuberculosis-infected individuals with or without Ss co-infection. Principal component analysis (PCA) was performed using cytokine concentrations and Mycobacterial Growth Inhibition Assay (MGIA) values from tuberculosis-infected (TBI) individuals with helminth co-infection (TBI⁺+Hel⁺) and without (TBI). The first two principal components (PC1: 28.6%; PC2: 16%) together explained 44.6% of the total variance. The PCA biplot shows distinct clustering of TBI and TBI⁺+Hel⁺ groups, indicating helminth-induced immune divergence. Cytokines associated with Th2/regulatory responses (IL-4, IL-5, IL-10, IL-13) clustered positively along PC1, aligning with MGIA values, while Th1/Th17-related cytokines (IL-17, IFN-γ, IL-2, TNF-α, IL-1α, IL-1β) loaded negatively. The segregation suggests that helminth infection reprograms cytokine networks from Th1/Th17-dominant toward Th2/regulatory-dominant profiles, consistent with reduced mycobacterial growth control.

3.7 Relationship between the levels of MGIA and cytokines

Correlation analysis between cytokine profiles and Mycobacterial Growth Inhibition Assay (MGIA) responses in tuberculosis-infected (TBI) individuals with Ss co-infection (TBI⁺Hel⁺) revealed distinct immunological associations. As shown in Table 2 3II, among the measured cytokines, IL-17 (r = –0.3884, p = 0.0110), IL-1α (r = –0.3537, p = 0.0371), and IL-1β (r = –0.3950, p = 0.0189) displayed significant negative correlations with MGIA, suggesting that elevated levels of these proinflammatory mediators were associated with reduced mycobacterial growth control. Other Th1 cytokines such as IFN-γ, IL-2, and TNF-α showed negative but non-significant trends with MGIA, while Th2 and regulatory cytokines (IL-4, IL-5, IL-10, IL-13) showed weak positive correlations, none reaching significance. Although some correlations reached statistical significance, the Spearman rho values were low, indicating that these associations are relatively weak and should be interpreted with caution. To further evaluate the independent contributions of cytokines and Ss-specific IgG to mycobacterial growth inhibition, all measured cytokines along with Ss-IgG were included in a multivariate regression model (Table 3). The model demonstrated that Ss-IgG, IL-17, IL-1α, and IL-1β remained negatively associated with MGIA values, while selected Type 2 cytokines such as IL-5, IL-10, and IL-13 showed positive associations. These results indicate that despite the presence of inflammatory mediators, the functional ability to restrict mycobacterial growth is impaired in Ss -TBI co-infected individuals, pointing toward a qualitative imbalance rather than quantitative deficiency in cytokine responses.

Table 2

Table 2. Relationship between the levels of MGIA and cytokines.

Table 3

Table 3. Multivariate regression model including Ss-IgG values.

4 Discussion

Our study demonstrates that helminth co-infection significantly compromises protective immune responses in individuals with tuberculosis infection (TBI). Our findings describe immune modulation associated specifically with Strongyloides stercoralis co-infection, as this was the only helminth species detected in the study population. Using a mycobacterial growth inhibition assay (MGIA), we observed impaired control of Mycobacterium tuberculosis (M.tb) H37Rv in vitro among TBI- Ss co-infected individuals compared to TBI+ individuals. This functional deficiency coincided with a shift in cytokine responses, characterized by reduced Th1, Th17, and innate proinflammatory cytokines, and enhanced Th2 and regulatory cytokine production.

Our data reveal that Ss co-infection in tuberculosis-infected individuals significantly alters hematological parameters. Although age, gender, and BMI were comparable between groups, TBI+ Hel+ individuals showed reduced hemoglobin and RBC levels, consistent with helminth-induced anemia due to chronic blood loss and iron deficiency (15). The decrease in lymphocyte counts may reflect helminth-driven immune modulation, where a shift from Th1 to Th2 responses suppresses cell-mediated immunity essential for controlling Mycobacterium tuberculosis (16).

Conversely, eosinophil levels were significantly elevated in TBI+ Hel+ individuals, a hallmark of helminth infections. Eosinophilia is driven by IL-5 and other Th2 cytokines, which promote eosinophil proliferation and survival (17). Additionally, helminth-induced Th2 polarization can dampen protective Th1 responses against TB, potentially impairing host defense mechanisms (18). These data emphasize the importance of considering helminth co-infection in TB-endemic regions, as it may influence disease progression, immune response, and treatment outcomes.

Our data demonstrates that in individuals with tuberculosis infection (TBI), coexisting Strongyloides stercoralis infection has been associated with a significant decrease in antigen-specific type 1, type 17, and pro-inflammatory cytokine responses. While mitogen-induced responses remained unchanged. As expected, PHA stimulation did not reproduce the differences observed with TB antigen stimulation, likely because mitogenic activation bypasses antigen-specific pathways that are more susceptible to helminth-associated modulation. No additional antigenic stimuli were evaluated beyond QFT TB1/TB2 peptides and H37Rv lysate. Cytokine responses, while reduced overall in the Ss infected group, varied depending on whether stimulation was performed with QFT antigens or H37Rv.

The stronger group differences observed with TBAg2 likely reflect the broader CD4⁺ and CD8⁺ T-cell stimulation captured by the TB2 tube, which may be more sensitive to Ss associated immune suppression, including effects on IL-1 family cytokines. Decreased IL-1α and IL-1β indicate weakened innate inflammatory signaling, while decreased Th1 and Th17 cytokines emphasize compromised macrophage activation and early mucosal defense. Th1 cytokines such as IFN-γ, IL-2, and TNF-α are critical for macrophage activation and granuloma maintenance, playing indispensable roles in controlling M.tb replication and dissemination (19) (20) (21). The observed reduction in these cytokines in helminth-infected individuals aligns with earlier reports suggesting helminth-driven suppression of Th1 responses (22, 23). Additionally, IL-17 and GM-CSF, both important for neutrophil recruitment and epithelial defense (24, 25), were significantly lower in helminth-co-infected individuals, suggesting impaired mucosal barrier integrity and granuloma function. Our findings align with those of Anwar et al. (26), who also reported reduced mycobacterial growth inhibition in individuals with predominantly Strongyloides stercoralis infection. In contrast, Shea et al. (27) observed improved MGIA in individuals with hookworm infection, which reverted to control levels following anthelmintic treatment. These contrasting results suggest that different helminth species may exert distinct effects on antimycobacterial immunity, and that helminth–TB interactions are not uniform across infections.

Innate cytokines such as IL-1α, IL-1β, IL-6, IL-12p70, and IL-18 were also significantly reduced. These cytokines initiate and amplify early immune responses and are essential for bridging innate and adaptive immunity (28, 29). Suppression of these mediators by helminths may delay or dampen the immune priming necessary for robust anti-TB responses. In contrast, we observed elevated IL-4, IL-5, IL-10, and IL-13 in the helminth-infected group, consistent with a Th2/regulatory bias. Such cytokines promote tissue repair and immune regulation but can inhibit effective antimicrobial activity (30). Notably, IL-10 has been associated with TB reactivation and persistence due to its suppressive effect on macrophage and T-cell function (31). Recent studies further confirm that helminth infection induces a Th2/regulatory environment, characterized by increased IL-10 and impaired Th1/Th17 responses, which can compromise anti-mycobacterial immunity (5).

Our data extends our earlier finding that Ss infection exacerbates tuberculosis infection (TBI) by revealing that helminth co-infection is associated with both qualitative alterations in cytokine–MGIA correlations and distinct immunological clustering by PCA. The significant negative associations between IL-17, IL-1α, and IL-1β and MGIA responses suggest that heightened inflammatory signaling is not protective but instead reflects immune dysregulation, leading to reduced bacterial control. Helminth infections are well known to induce Th2 and regulatory immune polarization, characterized by increased IL-4, IL-5, IL-10, and IL-13 production and expansion of T regulatory cells, which suppress Th1/Th17 effector responses critical for antimycobacterial immunity (23, 32, 36). The PCA-supported shift toward a Th2/regulatory cytokine signature further indicates that helminth co-infection reprograms immune homeostasis, promoting a milieu that favors chronic tolerance rather than pathogen clearance. Although IL-10 and IL-13 showed weak positive associations with MGIA, these likely represent compensatory anti-inflammatory feedback mechanisms that inadvertently dampen effective Th1-mediated protection. Collectively, these findings demonstrate that Ss infection skews host immunity away from protective Th1/Th17 balance, resulting in impaired functional control of Mycobacterium tuberculosis. This immune reprogramming may partly explain reduced vaccine responsiveness and poor TB outcomes in helminth-endemic settings (33–35). Multivariate model confirmed that Ss driven biomarkers such as Ss IgG, IL-17, IL-1α, and IL-1β contributed independently to impaired mycobacterial growth control, while elevated Type 2 cytokines (IL-5, IL-10, IL-13) showed positive associations with MGIA, further supporting a Ss associated shift away from protective Th1/Th17 pathways.

Our study has certain limitations. The sample size is relatively small. Furthermore, a causal relationship between Ss co-infection and compromised Mycobacterium tuberculosis-specific immunity cannot be inferred due to the cross-sectional design. Participants were not categorized by helminth species, burden, infection severity, or treatment history—all of which are known to affect immune modulation. TBI controls were not included because the study focused on TBI cases, and TBI–Ss+ individuals were rare in this population. Only the mycobacterial growth inhibition assay was used, without the use of complementary cellular assays like T-cell phenotyping or intracellular cytokine staining, and cytokine responses were evaluated at a single time point, which might not adequately capture the dynamic nature of immune responses.

Our study shows that shifting responses from proinflammatory Th1/Th17 to Th2/regulatory pathways, helminth co-infection reduces TB-specific immunity in TBI patients. These immune alterations may influence the performance of TB diagnostics and vaccines in helminth-endemic areas. Our findings emphasize the necessity of taking helminth burden into consideration when developing TB control strategies. They also highlight the significance of integrated intervention approaches and the need for further research to elucidate the mechanistic pathways that connect helminth infection, immune modulation, and TB risk.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement

Ethical approval was obtained from the Institutional Review Boards of the National Institute of Allergy and Infectious Diseases (NIAID), USA, and the National Institute for Research in Tuberculosis (NIRT), India (NCT04813328, NCT04526613, NIRT-IEC 2020 033 and NIRT-IEC 2020 005). Written informed consent was obtained from all study participants. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Author contributions

AR: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review and editing. BD: Investigation, Methodology, and Writing – review and editing. AP: Investigation, Methodology and Writing – review and editing. SM: Investigation, Methodology and Writing – review and editing. SS: Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – review and editing. SB: Conceptualization, Funding acquisition, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review and editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the Division of Intramural Research, the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.

Acknowledgments

We thank Dr. M. Satiswaran, Dr. Sushmitha, and Dr. B. Suganthi and the staff members of the Department of Epidemiology, NIRT, Chennai, for their valuable assistance in recruiting the patients for this study. We thank the staff members of the Department of Bacteriology, NIRT for their invaluable support and guidance in this study.

Conflict of interest

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

1. McSorley HJ and Maizels RM. Helminth infections and host immune regulation. Clin Microbiol Rev. (2012) 25:585–608. doi: 10.1128/CMR.05040-11

PubMed Abstract | Crossref Full Text | Google Scholar

2. Kudryavtsev I, Starshinova A, Rubinstein A, Kulpina A, Ling H, Zhuang M, et al. The role of the immune response in developing tuberculosis infection: from latent infection to active tuberculosis. Front Tuberculosis. 2024-September-30;Volume. (2024) 2(2024). doi: 10.3389/ftubr.2024.1438406

Crossref Full Text | Google Scholar

3. Mandal S, Bhatia V, Bhargava A, Rijal S, and Arinaminpathy N. The potential impact on tuberculosis of interventions to reduce undernutrition in the WHO South-East Asian Region: a modelling analysis. Lancet Reg Health Southeast Asia. (2024) 31:100423. doi: 10.1016/j.lansea.2024.100423

PubMed Abstract | Crossref Full Text | Google Scholar

4. Babu S and Nutman TB. Helminth-tuberculosis co-infection: an immunologic perspective. Trends Immunol. (2016) 37:597–607. doi: 10.1016/j.it.2016.07.005

PubMed Abstract | Crossref Full Text | Google Scholar

5. Chin KL, Fonte L, Lim BH, Sarmiento ME, and Acosta A. Immunomodulation resulting of helminth infection could be an opportunity for immunization against tuberculosis and mucosal pathogens. Front Immunol. (2023) 14:1091352. doi: 10.3389/fimmu.2023.1091352

PubMed Abstract | Crossref Full Text | Google Scholar

6. Garrido-Amaro C, Cardona P, Gasso D, Arias L, Velarde R, Tvarijonativiciute A, et al. Protective effect of intestinal helminthiasis against tuberculosis progression is abrogated by intermittent food deprivation. Front Immunol. (2021) 12:627638. doi: 10.3389/fimmu.2021.627638

PubMed Abstract | Crossref Full Text | Google Scholar

7. Painter H, Harriss E, Fletcher HA, McShane H, and Tanner R. Development and application of the direct mycobacterial growth inhibition assay: a systematic review. Front Immunol. (2024) 15:1355983. doi: 10.3389/fimmu.2024.1355983

PubMed Abstract | Crossref Full Text | Google Scholar

8. Bhengu KN, Naidoo P, Singh R, Mpaka-Mbatha MN, Nembe N, Duma Z, et al. Immunological interactions between intestinal helminth infections and tuberculosis. Diagnostics. (2022) 12:2676. doi: 10.3390/diagnostics12112676

PubMed Abstract | Crossref Full Text | Google Scholar

9. Babu S, Bhat SQ, Kumar NP, Kumaraswami V, and Nutman TB. Regulatory T cells modulate Th17 responses in patients with positive tuberculin skin test results. J Infect Dis. (2010) 201:20–31. doi: 10.1086/648735

PubMed Abstract | Crossref Full Text | Google Scholar

10. Chatterjee S and Nutman TB. Helminth-induced immune regulation: implications for immune responses to tuberculosis. PloS Pathog. (2015) 11:e1004582. doi: 10.1371/journal.ppat.1004582

PubMed Abstract | Crossref Full Text | Google Scholar

11. Rajamanickam A, Munisankar S, Bhootra Y, Dolla CK, Nutman TB, and Babu S. Coexistent helminth infection-mediated modulation of chemokine responses in latent tuberculosis. J Immunol. (2019) 202:1494–500. doi: 10.4049/jimmunol.1801190

PubMed Abstract | Crossref Full Text | Google Scholar

12. Bisoffi Z, Buonfrate D, Sequi M, Mejia R, Cimino RO, Krolewiecki AJ, et al. Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection. PloS Negl Trop Dis. (2014) 8:e2640. doi: 10.1371/journal.pntd.0002640

PubMed Abstract | Crossref Full Text | Google Scholar

13. Buonfrate D, Formenti F, Perandin F, and Bisoffi Z. Novel approaches to the diagnosis of Strongyloides stercoralis infection. Clin Microbiol Infect. (2015) 21:543–52. doi: 10.1016/j.cmi.2015.04.001

PubMed Abstract | Crossref Full Text | Google Scholar

14. Sato Y, Kobayashi J, Toma H, and Shiroma Y. Efficacy of stool examination for detection of Strongyloides infection. Am J Trop Med Hyg. (1995) 53:248–50. doi: 10.4269/ajtmh.1995.53.248

PubMed Abstract | Crossref Full Text | Google Scholar

15. Erin Logan AC and Horsnell WG. How helminths alter immunity to infection-the role of antibody in parasitic helminth infections. New York, NY: Springer (2014).

Google Scholar

16. Carabalí-Isajar ML, Rodríguez-Bejarano OH, Amado T, Patarroyo MA, Izquierdo MA, Lutz JR, et al. Clinical manifestations and immune response to tuberculosis. World J Microbiol Biotechnol. (2023) 39:206. doi: 10.1007/s11274-023-03636-x

PubMed Abstract | Crossref Full Text | Google Scholar

17. Mitre E and Klion AD. Eosinophils and helminth infection: protective or pathogenic? Semin Immunopathology. (2021) 43:363–81. doi: 10.1007/s00281-021-00870-z

PubMed Abstract | Crossref Full Text | Google Scholar

18. du Plessis N and Walzl G. Helminth-M. tb co-infection. How helminths alter Immun to infection. Adv Exp Med Biol.. (2014) 828:49–74. doi: 10.1007/978-1-4939-1489-0_3

PubMed Abstract | Crossref Full Text | Google Scholar

19. Maggioli MF, Palmer MV, Thacker TC, Vordermeier HM, McGill JL, Whelan AO, et al. Increased TNF-alpha/IFN-gamma/IL-2 and decreased TNF-alpha/IFN-gamma production by central memory T cells are associated with protective responses against bovine tuberculosis following BCG vaccination. Front Immunol. (2016) 7:421. doi: 10.3389/fimmu.2016.00421

PubMed Abstract | Crossref Full Text | Google Scholar

20. Hickman SP, Chan J, and Salgame P. Mycobacterium tuberculosis induces differential cytokine production from dendritic cells and macrophages with divergent effects on naive T cell polarization. J Immunol. (2002) 168:4636–42. Available online at: https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/tb-reports/global-tuberculosis-report-2024. 2024. https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/tb-reports/global-tuberculosis-report-2024 (Accessed on December 2, 2025).

PubMed Abstract | Google Scholar

21. Cardona P and Cardona PJ. Regulatory T cells in mycobacterium tuberculosis infection. Front Immunol. (2019) 10:2139. doi: 10.3389/fimmu.2019.02139

PubMed Abstract | Crossref Full Text | Google Scholar

22. George PJ, Kumar NP, Jaganathan J, Dolla C, Kumaran P, Nair D, et al. Modulation of pro-and anti-inflammatory cytokines in active and latent tuberculosis by coexistent Strongyloides stercoralis infection. Tuberculosis. (2015) 95:822–8. doi: 10.1016/j.tube.2015.09.009

PubMed Abstract | Crossref Full Text | Google Scholar

23. Bewket G, Kiflie A, Tajebe F, Abate E, Schon T, and Blomgran R. Helminth species dependent effects on Th1 and Th17 cytokines in active tuberculosis patients and healthy community controls. PloS Negl Trop Dis. (2022) 16:e0010721. doi: 10.1371/journal.pntd.0010721

PubMed Abstract | Crossref Full Text | Google Scholar

24. Fan X, Zhao X, Wang R, Li M, Luan X, Wang R, et al. A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses. Front Immunol. (2023) 14. doi: 10.3389/fimmu.2023.1276887

PubMed Abstract | Crossref Full Text | Google Scholar

25. Khader SA, Bell GK, Pearl JE, Fountain JJ, Rangel-Moreno J, Cilley GE, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol. (2007) 8:369–77. doi: 10.1038/ni1449

PubMed Abstract | Crossref Full Text | Google Scholar

26. Anwar S, Turienzo CF, Tsang L, Smith SG, Fletcher H, Toulza F, et al. Impact of helminth co-infection and treatment on mycobacterial growth inhibition in UK migrants with TB infection. IJTLD Open. (2025) 2:217–23. doi: 10.5588/ijtldopen.24.0528

PubMed Abstract | Crossref Full Text | Google Scholar

27. O’Shea MK, Fletcher TE, Muller J, Tanner R, Matsumiya M, Bailey JW, et al. Human hookworm infection enhances mycobacterial growth inhibition and associates with reduced risk of tuberculosis infection. Front Immunol. (2018) 9:2893. doi: 10.3389/fimmu.2018.02893

PubMed Abstract | Crossref Full Text | Google Scholar

28. Mayer-Barber KD, Andrade BB, Oland SD, Amaral EP, Barber DL, Gonzales J, et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature. (2014) 511:99–103. doi: 10.1038/nature13489

PubMed Abstract | Crossref Full Text | Google Scholar

29. Van Den Eeckhout B, Tavernier J, and Gerlo S. Interleukin-1 as innate mediator of T cell immunity. Front Immunol. (2020) 11:621931. doi: 10.3389/fimmu.2020.621931

PubMed Abstract | Crossref Full Text | Google Scholar

30. Maizels RM and Yazdanbakhsh M. Immune Regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol. (2003) 3:733–44. doi: 10.1038/nri1183

PubMed Abstract | Crossref Full Text | Google Scholar

31. Redford PS, Murray PJ, and O’Garra A. The role of IL-10 in immune regulation during M. tuberculosis infection. Mucosal Immunol. (2011) 4:261–70. doi: 10.1038/mi.2011.7

PubMed Abstract | Crossref Full Text | Google Scholar

32. Schick J, Altunay M, Lacorcia M, Marschner N, Westermann S, Schluckebier J, et al. IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity. Elife. (2023) 12:e72923. doi: 10.7554/eLife.72923

PubMed Abstract | Crossref Full Text | Google Scholar

33. Loke P, Lee SC, and Oyesola OO. Effects of helminths on the human immune response and the microbiome. Mucosal Immunol. (2022) 15:1224–33. doi: 10.1038/s41385-022-00532-9

PubMed Abstract | Crossref Full Text | Google Scholar

34. Vacca F and Le Gros G. Tissue-specific immunity in helminth infections. Mucosal Immunol. (2022) 15:1212–23. doi: 10.1038/s41385-022-00531-w

PubMed Abstract | Crossref Full Text | Google Scholar

35. Webster HC, Gamino V, Andrusaite AT, Ridgewell OJ, McCowan J, Shergold AL, et al. Tissue-based IL-10 signalling in helminth infection limits IFNγ expression and promotes the intestinal Th2 response. Mucosal Immunol. (2022) 15:1257–69. doi: 10.1038/s41385-022-00513-y

PubMed Abstract | Crossref Full Text | Google Scholar

36. Rajamanickam A, Munisankar S, Dolla C, Nutman TB, and Babu S. Cytotoxic T- lymphocyte-associated antigen 4 (CTLA-4)-and programmed death 1 (PD-1)-mediated regulation of monofunctional and dual functional CD4+ and CD8+ T-cell responses in a chronic helminth infection. Infection Immun. (2019) 87:10.1128/iai00469–00419. doi: 10.1128/IAI.00469-19

PubMed Abstract | Crossref Full Text | Google Scholar