The association between type 2 diabetes and pulmonary cavitation revealed among IGRA-positive tuberculosis patients

The co-occurrence of tuberculosis (TB) and diabetes mellitus (DM) presents a significant obstacle to TB eradication. Pulmonary cavitation can occur in severe cases of TB, particularly in patients with DM. From 1 May 2014 through 30 June 2019, we conducted a cross-sectional study of 1,658 smear- or culture-confirmed pulmonary TB (PTB) patients at the Second Department of Pulmonary Medicine and Tuberculosis, Shenzhen, China. A total of 861 participants who satisfied the criteria (chest CT scan for cavitation, interferon-gamma release assay (IGRA), diagnosis of diabetes mellitus), with the median age of 36.7 years, 63.6% of male, 79.7% IGRA positive, 13.8% with diabetes, and 40.8% with pulmonary cavitation, were included in the study. The association between diabetes and pulmonary cavitation was confirmed in these TB patients (adjusted OR, 2.54; 95% CI, 1.66–3.94; p < 0.001). No associations were observed between diabetes and IGRA, as well as between lung cavitary and IGRA. Based on the criteria of IGRA+/–, pulmonary cavitation+/–, and DM+/–, the further analysis with univariate and multivariate logistic regression were conducted in six subgroups. The significant association between diabetes and pulmonary cavitation was further confirmed in the IGRA+ subgroup (adjusted OR, 3.07; 95% CI, 1.86–5.16; p < 0.001) but not observed in IGRA- individuals. This observation suggests that different immunological mechanisms of pulmonary cavitary/DM may be employed in IGRA+ TB patients from IGRA- TB patients.


Introduction
Tuberculosis (TB) remains a major global health challenge, particularly in low-and middle-income countries.According to the World Health Organization, there were 10.6 million new TB cases and 1.6 million deaths from TB in 2021 (1).China has the world's second largest population of TB patients, with approximately 1 million new TB cases reported each year.
Diabetes, as a metabolic disorder disease, is associated with innate and adaptive immune dysfunctions and alterations in specific cytokines and chemokines (2).Previous studies have reported a paradoxical hyperinflammatory response in TB patients with DM, such as IFN-γ, IL-2, and TNF-a (3)(4)(5).There were studies indicating that diabetes mellitus (DM) is associated with an increased risk of TB (6)(7)(8).It was reported that individuals with DM have a 2-to 4-fold higher risk of active TB, and up to 30% of individuals with TB are likely to have DM (9).As a key pathological feature and a dangerous consequence of clinical TB, pulmonary cavitation is associated with poor treatment outcomes, relapse, higher transmission rates, and the development of drug resistance (10).Previous studies proved that the incidence of cavitary TB is higher in diabetic patients compared to nondiabetic patients (11)(12)(13)(14).TB individuals with DM experience persistent hyperglycemia, leading to a compilation of aberrant metabolic changes and increased superoxide production, which activates inflammatory pathways and leads to immune system dysfunction, indicating an abnormal and progressive immune response that favors cavitation in diabetic TB individuals (15).
Interferon-γ serves as a crucial lymphokine for the protective immune response to M. tuberculosis (16,17).Reduced sensitivity of IFN-γ release has been demonstrated in humans with physiologically and pathologically immunocompromised factors (18).The interferon-γ release assay (IGRA), measuring early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) to indicate a specific cellular immune response to M. tuberculosis, has been routinely used in clinical screening for TB infection.Additionally, it serves as an adjunct diagnostic biomarker for active TB (19).In this study, we assessed whether cavitary or DM is associated with MTB-stimulated interferon-γ secretion in clinical TB patients.

Materials and methods . Ethics statement
This study was approved by the ethics committee of the Third People's Hospital of Shenzhen (IRB No.: 2021-014-02).All patients' private information was deleted, and each case was coded with a pathology accession number to protect patient privacy.The ethics committee waived the requirement for written informed consent from patients.The study was conducted in accordance with the ethical standards and confidentiality principles outlined in the Helsinki Declaration.

. Study population
From 1 May 2014 to 30 June 2019, a total of 1,658 patients with microbiologically confirmed pulmonary TB were recruited in the Second Department of Pulmonary Medicine and Tuberculosis, the Third People's Hospital of Shenzhen.A positive sputum smear or culture was indicative of microbiologically confirmed pulmonary TB, excluding non-mycobacterium pulmonary diseases.Sociodemographic characteristics, outpatient characteristics, outpatient and inpatient clinical encounters, medication prescriptions and fills, medical conditions, procedures, and laboratory results.Among the 1,658 patients, the exclusion criteria for this study included (1) absence of IGRA results (n = 236); (2) lack of CT images (n = 552); (3) onset of the rheumatic disease (n = 6); and (4) absence of basic information (n = 3).Ultimately, 861 patients were enrolled in this study (Figure 1).

. Methods
The enzyme-linked immune-spot tuberculosis (ELISPOT.TB) assay was used for screening the interferon-gamma release.Blood glucose and glycated hemoglobin (HbA1c) tests along with chest high-resolution computed tomography (CT) were performed at the patient's first admission.The IGRA assay was performed according to previously published research methods (20) and briefly described as follows: Panel A (peptides of ESAT-6 aa 21 to 40, aa 51 to 70, and aa 71 to 90) and Panel B (peptides of CFP-10 aa 21 to 40, aa 51 to 70, and aa 66 to 85) were used as antigens at a concentration of 10 µg/ml.Peripheral blood mononuclear cells (PBMCs) were freshly isolated from 10 ml of anticoagulated blood samples using the Ficoll density gradient centrifugation method.A total of 2 × 10 5 cells/well were seeded in duplicate in 96-well plates pre-coated with an anti-IFN-capture monoclonal antibody.The cells were then stimulated with different antigens for 24 h at 37 • C, 5% CO 2 .PBMCs in medium alone or stimulated with phytohemagglutinin were used as negative or positive controls.Biotinylated anti-IFNdetection monoclonal antibody was added for 4 h, followed by the addition of streptavidin-alkaline phosphatase conjugate for 1 h.Subsequently, nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3indolylphosphate) chromogenic substrate was added for staining.The spots were counted using BioReader 4,000 Pro-X (Biosys, Germany).The test result of ELISPOT.TB assay was considered positive if either or both of Panel A had 18 or Panel B had 11 or more spots than the negative control.
Diabetes mellitus was defined according to the diagnostic criteria established by the WHO, and patients with HbA1c ≥ 6.5% were commonly diagnosed with DM (21).The baseline information on demographic variables (for e.g., sex, age, ethnicity, marital status, and work status), behavioral risk factors (for e.g., smoking and alcohol use) and paraclinical data [for e.g., hemoglobin (HGB)] along with serum albumin (ALB), white blood cell count (WBC), lymphocyte count (LYN), neutrophilic granulocyte count (GRA), monocyte count (MONO), and Creactive protein (CRP) were considered as confounders.The CT scanners used in this study included CT64 (China), Light Speed 16 and General Electric (GE) Revolution CT256 from United Imaging.The machine (Toshiba Asteion; Toshiba, Tokyo, Japan) parameters were as follows: 1.15-mm section thickness, 3-mm gap, 1-or 2-s scanning time per section, 120 kV, and 200 mA.The images were photographed at the lungs (window width, 1,800 HU, window level, 400 HU).Two experienced radiologists reviewed the CT images and reached a consensus decision at the Third People's Hospital of Shenzhen.The definition of pulmonary cavitation can be referred in this article (3), and a representative CT image is displayed in Supplementary Figure 1. .

Statistical analysis
The study data were entered into a spreadsheet and analyzed using the statistical software R (version 4.1.2)(22).Bioinformatic analysis was performed using the OmicStudio tools at https://www.omicstudio.cn.
Bivariate associations were analyzed using chi-square tests for categorical variables and the Wilcoxon rank sum or Kruskal-Wallis tests for continuous variables.A two-sided p-value < 0.05 was considered statistically significant.To identify the risk factors associated with the presence of pulmonary cavitation in TB patients, univariate and multivariate logistic regression models were developed.The baseline information on demographic variables (for e.g., sex, age, ethnicity, marital status, and work status), behavioral risk factors (for e.g., smoking and alcohol use), and paraclinical data (for e.g., HGB, ALB, WBC, LYN, GRA, MONO, and CRP) were considered as confounders.The odds ratios (ORs) and their 95% confidence intervals (95% CIs) were also calculated to estimate the degree of association between different variables and pulmonary cavitation.Variables with a p-value ≤ 0.20 on the univariate analysis were initially offered to a saturated multivariate logistic regression model.The optimized model was generated using a "both" stepwise process, and the most appropriate model was determined by assessing the likelihood test and the minimum Akaike information criterion (AIC) value (23).The variance inflation factor (VIF) was used to detect multicollinearity in the regression analysis.A variable with VIF >5 was considered to have multicollinearity, and variables with multicollinearity were manually removed step by step until it was eliminated.The model was further assessed using the Hosmer-Lemeshow goodness-of-fit test.A receiver operating characteristic (ROC) curve was produced to display the predictive accuracy of the model using the R package "pROC."The "lme4" and "gtsummary" packages were used to build and display the univariate and multivariate logistic regression models (24).The "tidyverse" package was used for data import, manipulation, and visualization (25).Additionally, we adopted a similar approach as above to explore the potential association of predictors with IGRA and the relationship between predictors and pulmonary cavitation depending on the IGRA results.

. Univariate and multivariate logistic regression analyses of risk factors in the specific subgroup of TB patients
According to the pulmonary cavitation+/-, diabetes+/-, and IGRA+/-, we categorized the enrolled TB patients into six specific subgroups and further conducted the risk factors analysis.

Discussion
The univariate and multivariate logistic regression models have been established for data analysis in this project.As shown in Table 2, diabetes was significantly associated with pulmonary cavitation in the univariate analysis (OR, 3.27; 95% CI, 2.19-4.94;p < 0.001), and this association was not significant in the multivariate regression analysis.Additionally, in Table 1, the significant association between cavitary and diabetes was identified in the TB group (adjusted OR, 2.54; 95% CI, 1.66-3.94;p < 0.001).An ROC curve was generated to assess the predictive value of the constructed model.or culture-positive, clinical, and 5-year population-based crosssectional study, the investigation of the association between clinical characteristics and pulmonary cavitation among TB patients was conducted.We found consistent evidence for an increased risk of cavitary TB with diabetes.Additionally, the significant association between diabetes and cavitary has been narrowed to "IGRA positive" TB patients rather than "IGRA negative" TB patients.
Hyperglycemia significantly affected the presentation of radiographic manifestations and was associated with severe TB (26).The possible underlying mechanism was considered to be an impaired immune function in patients with diabetes.IFN-γ, TNF-α, IL-17, and IL-23 play crucial roles in the induction and maintenance of protective immune responses against TB (27).Accumulating evidence has shown that IFN-γ is responsible for driving cell-mediated immune responses through production by Th1 cells and has regulatory properties by acting as an inducer of Th2 responses.Using chronic M.tb infection in hyperglycemic mice, adaptive immunity was found to be delayed, as shown by reduced early production of IFN-γ in the lungs and by the presence of fewer M.tb antigen (ESAT-6)-responsive T cells (28).The animal data have been supported by two clinical studies from Tsukaguchi et al. (4,29).Furthermore, using a model of in vitro granulomas generated by DM2 patients' peripheral blood mononuclear cells infected with M. bovis BCG, the authors observed that PBMCs from GSH-supplemented patients produced enhanced levels of IFN-γ and controlled bacterial replication more efficiently than those from placebo-treated patients (30).In particular, the levels of IFN-γ were markedly reduced in samples from patients with poor glycemic control, which also failed to inhibit bacterial growth (31).Thus, the different immune responses involved in IFN-γ between TB with DM and without DM may be due not only to differences in the frequencies of innate and adaptive immune cells but also to uncontrolled hyperglycaemia.Proinflammatory responses in the lungs can lead to tissue damage, disrupting normal tissue architecture and consequently  (39) reported that IFN-γ released from CD8+ T cells downregulated the expression of SLC3A2 and SLC7A11 and promoted tumor cell lipid peroxidation and ferroptosis.In addition, Orvedahl et al. (40) identified autophagy genes as central mediators of myeloid cell survival to IFN-γ mediated by TNF signaling via receptor interacting protein kinase 1 (RIPK1)and caspase 8-mediated cell death.Therefore, the IFN-γ activity involved in various forms of cell death suggests that IFN-γ may improve a complex phenotype, such as "tissue damage, " by modulating cell death.IGRA detects IFN-γ released by specific T cells, reflecting the TB lymphocyte function and the host immune state to TB. Patients with severe TB, such as military TB and tuberculous meningitis, have been proven to have lower CD4 T-cell counts and impaired T-cell function.In our study, we retrospectively included 861 microbiological PTB participants for analysis.And the association between type 2 diabetes and pulmonary cavitation was found only in IGRA-positive TB patients.
Our observation indicates that IFN-γ plays a crucial role in the development of cavitary with diabetes in TB patients.
The study had several limitations that should be considered.First, it was conducted in a specialized TB hospital with smearor culture-positive confirmed TB patients, which resulted in a significantly lower number of negative IGRA patients (175) compared to positive IGRA patients (686).This retrospective study was based on the records of 5 years of clinical practice that were collected, indicating that further prospective cohort studies and follow-up studies are indispensable.Second, there were unexpected pathological changes in the enrolled TB patients, including the onset of infection, progression stages, and relapse.Addressing the phenomena between the different stages of M.tb infection and IFNγ-related immunological regulation requires further evaluation with diabetic and latent TB animal models.Nevertheless, the population in the present study was an unbiased group of smear or culture-confirmed TB patients.Therefore, we believe that our results are represents more real-world outcomes.
In conclusion, in this study we found that there was a significant association between pulmonary cavitation and type 2 diabetes among the IGRA positive TB patients.This observation strongly suggests that different immunological mechanisms of pulmonary cavitary/DM would be employed in IGRA+ TB and IGRA-TB individuals.Further exploration of pulmonary cavitary/DM in IGRA+ and IGRA-TB patients would reveal more immunological characteristics of the tuberculosis disease.
TABLE Univariate and multivariate logistic regression analyses of risk factors for pulmonary cavitation in TB patients (n =).
TABLE Univariate and multivariate logistic regression analyses of risk factors for type diabetes in TB patients (n =).
The model exhibited intermediate accuracy with an area under the ROC curve (AUC) of 0.706, indicating the high accuracy of this model.In this smear TABLE Univariate and multivariate logistic regression analyses of risk factors for IGRA in TB patients (n =).
TABLE Univariate and multivariate logistic regression analyses of risk factors for pulmonary cavitation in TB patients who tested IGRA positive (n =).
TABLE Univariate and multivariate logistic regression analyses of risk factors for type diabetes in TB patients who tested IGRA positive (n = ).TABLE Univariate and multivariate logistic regression analyses of risk factors for pulmonary cavitation in TB patients who tested IGRA negative (n = ).In vivo, in mice bearing ovarian tumors, Wang et al.