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Although the biological basis for the increased susceptibility of diabetic patients to tuberculosis remains unclear, the world is undergoing a type 2 diabetes pandemic. We hypothesize that chronic hyperglycemia leads to immunocompromise that facilitates progression to active tuberculosis. To assess this possibility, we determined whether patients with tuberculosis and diabetes (particularly those with chronic hyperglycemia), compared with patients with tuberculosis who did not have diabetes, presented altered cytokine responses to a mycobacterial antigen.
Samples of whole blood from patients with tuberculosis and diabetes and from patients with tuberculosis who did not have diabetes was stimulated in vitro with purified protein derivative from Mycobacterium tuberculosis. We then determined whether there was an association between the levels of innate and adaptive cytokines secreted in response to the antigen and diabetes status, or diabetes with chronic hyperglycemia (measured by glycosylated hemoglobin level), after controlling for possible confounders.
Innate and type 1 cytokine responses were significantly higher in patients with tuberculosis who had diabetes than in nondiabetic control subjects. The effect was consistently and significantly more marked in diabetic patients with chronic hyperglycemia.
These data provide preliminary evidence that type 2 diabetes, especially type 2 diabetes involving chronic hyperglycemia, is associated with an altered immune response to M. tuberculosis. More-detailed knowledge of the underlying mechanisms should focus on the effect of chronic hyperglycemia on the immune response to help in understanding the enhanced susceptibility of diabetic patients to tuberculosis.
One of the many consequences of the pandemic of type 2 diabetes  is an increased frequency and severity of infection in diabetic patients, including up to an 8-fold increase in the risk of developing pulmonary tuberculosis . The molecular basis for this susceptibility to tuberculosis is unclear. One possibility is a compromised immune response in diabetic patients that facilitates either primary progressive tuberculosis or reactivation of a latent tuberculosis infection . Studies evaluating immunity to other microbial antigens in diabetic patients suggest that adaptive and innate responses are compromised, particularly in patients with chronic hyperglycemia (measured by elevated glycosylated hemoglobin [HbA1c] level) [4–15]. Immune impairments appear to be reversible with improved glycemic control [10, 16, 17].
Although susceptibility to tuberculosis in diabetic patients has been recognized for more than a century, only now, when we are faced by the current type 2 diabetes epidemic, are we beginning to address the public health impact and potential mechanisms of this association. Cytokines of the innate and adaptive immune systems orchestrate the response to Mycobacterium tuberculosis, with subsequent induction of cellular type 1 immunity, which is critical for protection [18–23]. A recently developed mouse model for diabetes and tuberculosis revealed higher bacterial burden and altered expression of IFN-γ in mice with chronic, but not acute, diabetes . Persistent hyperglycemia apparently plays a significant role in the pathology.
In this context, we assessed differences in cytokine secretion in response to an M. tuberculosis antigen in diabetic and nondiabetic patients with tuberculosis, selected from a population of patients known to have elevated rates of type 2 diabetes [25, 26]. To do this, we adapted a relatively simple ex vivo stimulation procedure designed to screen for variations in innate and adaptive immune responses to M. tuberculosis antigens. We hypothesized that, if impairment of the immune response plays a role in susceptibility to tuberculosis in patients with diabetes, we should be able to detect differences in cytokine responses to M. tuberculosis antigens in such patients, particularly in those with poor glycemic control.
We identified patients with suspected pulmonary tuberculosis (age, ≥20 years) who presented sequentially at reference clinics for tuberculosis in southern Texas (Hidalgo and Cameron County Health Departments) and northeastern Mexico (Secretaria de Salud de Tamaulipas, Matamoros). Patients without microbiological confirmation of M. tuberculosis infection (i.e., without positive smear and/or culture results) were excluded from analysis, as were patients with ELISA-confirmed or self-reported HIV infection, because of the strong and well-characterized association of HIV infection with immune impairment and tuberculosis . We also excluded patients who had received treatment for >8 days, to avoid possible confounding by modulation of the immune response by treatment. We did include patients who had received antimycobacterial treatment for ≤8 days, after first establishing that they did not differ from untreated patients in their response to PPD (see Results). Participants were recruited in accordance with guidelines from the institutional review boards of the participating institutions in both countries.
Patient interviews documented sociodemographic characteristics, clinical findings, and self-reported diabetes. Height and weight were recorded to estimate body mass index (calculated as the weight in kilograms divided by height in meters squared). Whole blood in EDTA was stored at −20°C for measurement of HbA1c level. A heparin tube was used for whole blood cytokine assays (described below), and stored plasma specimens were used for determining insulin levels.
Quality of glycemic control over the preceding 3 months was assessed by measuring HbA1c level with use of a commercial kit standardized for blood samples that have been previously frozen (Glyco-Tek Affinity Column; Helena Laboratories). This kit defines an abnormal (elevated) level of HbA1c to be >6.2% of total hemoglobin .
A whole blood stimulation assay was adapted to evaluate cytokine secretion in response to an M. tuberculosis antigen (PPD; the experimental antigen [Mycos Research]), staphylococcal enterotoxin B (SEB; the positive control superantigen [Sigma-Al-drich]), or no antigen (NEG; cytokine background control) in the presence of costimulatory antibodies (CD28 and CD49d [BD Biosciences]; Appendix [online only]) [29, 30]. On obtaining the blood sample from each patient, one set of the antigen-antibody preparations (PPD, SEB, and NEG), each in a separate tube, was thawed so that 1 mL of blood per tube could be immediately added and mixed gently, for final concentrations of 10 μg/mL of SEB, 20 μg/mL of PPD, and 1 μg/mL of each antibody . After 22–24 h of incubation at 37°C in a waterbath, the supernatants were harvested and stored frozen in aliquots at −20°C. Quantification of IFN-γ, IL-1β, and IL-6 was performed with use of ELISA (OptEIA; BD Biosciences), and quantification of IL-2, IL-4, IL-8, IL-10, IL-12, IL-13, TNF-α, and granulocyte monocyte–CSF was performed with use of multiplex ELISA (LINCOplex kit; Millipore) in a Luminex 100 reader (Luminex).
Hyperglycemia (defined as a fasting glucose level ≥126 mg/dL or a random glucose level ≥200 mg/dL) was estimated using a hand-held glucometer (AccuCheck Advantage [Roche]) on EDTA-anticoagulated blood specimens. Patients with previously diagnosed diabetes (including patients with self-reported diabetes and/or patients receiving diabetes medication) or patients not reporting diabetes but with hyperglycemia at diagnosis were classified as having diabetes . In line with the current national guidelines  and taking into account patient age, we estimated that at least 95% of these patients had type 2 diabetes .
Data analysis was performed using SAS, version 9.1 (SAS). Cytokine responses to antigens were adjusted by subtracting the background level of each cytokine (NEG) from either SEB (adjusted SEB, defined as SEB minus NEG) or PPD (adjusted PPD, defined as PPD minus NEG). Univariate analysis was conducted by χ2 test (if n was >5 persons per group) or Fisher's exact test (if n was ≤5 persons per group) for dichotomous variables and by Kruskall-Wallis test for continuous variables. Multivariate linear models were built to test the association between cytokine levels (the dependent variable) and the main comparison groups (diabetes status and HbA1c level). Natural log transformation of the adjusted value for each cytokine in response to PPD was performed to create a normal distribution of cytokine levels prior to conducting multiple linear regression. P values <.05 were considered to be statistically significant.
Sixty-eight patients with tuberculosis met our inclusion criteria. Two patients with elevated HbA1c levels but no other evidence of diabetes were excluded. The characteristics of the remaining 66 patients are shown in table 1. Twenty-nine (44%) had diabetes, and of these, 19 (29% of all 66 patients; 66% of the 29 diabetic patients) had high HbA1c levels, indicating chronic hyperglycemia. Nearly all of these patients were nonblack Hispanics (Mexican Americans). We evaluated differences between the participants with respect to diabetes and HbA1c status, which were our variables of interest. There were marginal differences between the study groups with respect to sex and bacille Calmette-Guérin vaccination history. Otherwise, the groups did not differ with respect to sociodemographic characteristics and tuberculosis symptoms (including fever, chills, cough, and productive cough; data not shown). As expected, mean body mass index, HbA1c levels, insulin levels, and the rate of hyperglycemia were significantly higher in the group of patients with diabetes (compared with nondiabetes group) and in the group of patients with diabetes who had elevated HbA1c levels (compared with group of patients who had normal levels).
Stimulation with the superantigen SEB served as a positive control to ensure that lymphocyte stimulation conditions were appropriate. All participants had at least 1 cytokine level >1000 pg/mL in response to SEB (i.e., adjusted SEB), indicating that WBCs were viable and capable of responding to an antigenic stimulus (data not shown). The response to the experimental mycobacterial antigen PPD (i.e., adjusted PPD) was assessed in all participants by measuring cytokine levels. Throughout our study, cytokines were generally classified as innate (IL-1β, IL-6, TNF-α, IL-8, and granulocyte monocyte–CSF), adaptive type 1 (IL-12, IL-2, and IFN-γ), or adaptive type 2 (IL-4, IL-10, and IL-13) to provide an overall picture of the type of response in each of the study groups. However, we realize that this classification is an oversimplification of the complexity of the immune responses, with some cytokines participating in >1 type of response. More than 92% of the participants had a detectable response (cytokine level, >0 pg/mL) to all tested innate cytokines, as well as to IFN-γ, IL-2, and IL-10. In contrast, IL-12, IL-4, and IL-13 responses were only detected in 41%, 27%, and 71% of the participants, respectively (table A1; online only). Because the response to IL-12 and IL-4 was not measurable in >50% of the participants, and because there were no differences between the diabetes or HbA1c study groups (data not shown), these 2 cytokines were not evaluated further.
We also sought any other possible host factor that could have an effect on the induced cytokine levels. Among the host characteristics not related to diabetes (age, sex, country of patient enrollment, smear result, duration of treatment, and history of bacille Calmette-Guérin vaccination), country of enrollment was the characteristic most frequently associated with changes in expression of cytokines. Other nondiabetes host factors showed random associations with cytokine levels (table 2). In contrast, all of the characteristics associated with diabetes (presence of diabetes, HbA1c level, hyperglycemia, and body mass index) were frequently associated with cytokine expression and showed similar patterns of cytokine types (table 2). For example, expression levels of IFN-γ, IL-2, and IL-13 were associated not only with diabetes, but also with hyperglycemia, body mass index, and HbA1c level. Diabetes alone was associated with altered expression of fewer cytokines than was elevated HbA1c level. Altogether, these data suggest that type 2 diabetes is associated with the level and profile of cytokine responses to M. tuberculosis PPD.
We conducted further analysis using HbA1c because of its frequent association with cytokine expression (table 2), as well as its precision for estimating the level of diabetes control as a continuous variable. We show the relationship between HbA1c and cytokine expression levels in response to PPD by scatter plots in figure 1. The response of several cytokines to PPD was biphasic (data for TNF-α, IL-2, and IFN-γ are shown in figure 1A–C; data for IL-6, granulocyte monocyte–CSF, and IL-13 are not shown); that is, patients with tuberculosis who had HbA1c levels ≤6.2% of total hemoglobin showed a heterogeneous level of cytokine expression, whereas patients with tuberculosis who had elevated HbA1c levels were “high” cytokine producers. This pattern contrasted with the one observed for cytokine responses to PPD that did not show an association with elevated HbA1c level, such as that for IL-8 (figure 1D). Significant associations are shown in the Appendix (table A2; online only). Together, these data suggest that HbA1c level is associated with the level and profile of cytokine responses to M. tuberculosis PPD.
We conducted multiple linear regression models to establish whether diabetes and poor diabetes control (as indicated by HbA1c level) were associated with the response of each cytokine to PPD after controlling for potential confounding variables. The independent variables entered in the models included either diabetes or HbA1c level, depending on the model tested, together with age and sex, as well as any of the other host factors that were related to a given cytokine response (dependent variable). To insure that we did not miss any other potentially important variables in the final model, we used a P value ≤.10 by univariate analysis as our inclusion level (table 2). HbA1c level was evaluated as a continuous variable to obtain maximum analysis power. We then conducted a stepwise removal of independent variables with the highest P values, leaving in the final model either diabetes or HbA1c level (depending on the model) and any host characteristics with P ≤ .05. The response to PPD was significantly greater for cytokines promoting or directly mediating cell-mediated immunity, including TNF-α, granulocyte monocyte–CSF, IFN-γ, and IL-2 in patients with diabetes (compared with patients with no diabetes) or in patients with elevated HbA1c levels (compared with patients with normal HbA1c levels). Furthermore, patients with elevated HbA1c levels also expressed significantly higher IL-1β levels (table 3). Thus, the response to PPD in patients with diabetes or high HbA1c levels was up-regulated for innate and type 1 cytokines but not for type 2 cytokines.
The underlying biological explanation for the increased frequency of tuberculosis among patients with diabetes is not understood. We show that, among patients with tuberculosis, there are differences in the innate and cellular cytokine responses to stimulation with PPD from M. tuberculosis that are associated with diabetes. However, when we examined data from patients with diabetes who had poorly controlled blood glucose levels (determined on the basis of HbA1c level), the effect on cytokine levels was more striking and consistent, supporting our hypothesis that persistent hyperglycemia may play a key role in altering the immune responses to M. tuberculosis in diabetic patients. Further elucidation of the mechanisms underlying the relationship between tuberculosis and type 2 diabetes should focus on the effect of poor glycemic control on the immune response.
Interestingly, elevated HbA1c level consistently exhibited up-regulation of specific cytokines, suggesting a significant association between chronic hyperglycemia and the immune response to M. tuberculosis. To our knowledge, there is no other underlying condition described to date that displays such a marked association with the intensity of the cytokine response to a mycobacterial antigen.
The cytokines that were up-regulated in response to PPD in patients with diabetes or high HbA1c levels were associated with the innate and type 1 responses, but were not associated with type 2 responses. In contrast, IL-4 response was undetectable in >50% of the patients with tuberculosis who had or did not have diabetes (table A1; online only) but was detected in 94% of these same patients in response to SEB (data not shown). We did not find an association between up-regulation of type 1 or type 2 cytokines and blood insulin levels (data not shown). Thus, our findings differ with the reported Th2 polarization of lymphocytes after in vitro incubation with insulin , possibly because of technical differences. In the study cited above , lymphocytes were exposed in vitro to supraphysiologic levels of insulin (1.6 μU/mL) to mimic intensive insulin therapy, whereas our patient's elevated insulin levels are likely to be a response to chronic insulin resistance.
IFN-γ is a key Th1 signature cytokine that is up-regulated by M. tuberculosis and yet is also critical for tuberculosis control. In our study, patients with tuberculosis and elevated HbA1c levels had significantly more production of IFN-γ in response to PPD than did nondiabetic patients with tuberculosis. This finding may appear to be paradoxical, because patients with diabetes are more susceptible to tuberculosis. A possible explanation is that diabetic patients present alterations in the downstream signal transduction of key Th1 and innate immune response cytokines, possibly as the result of an increase in advanced glycation end products that can bind and modify protein function . This could lead to suppression of downstream responses that are critical for eliminating M. tuberculosis despite high levels of protective cytokines and/or accumulation of dysfunctional cytokines in plasma due to advanced glycation end products modification. The latter may result from failure of cytokines to induce negative feedback regulators (e.g., suppressor of cytokine signaling) that normally control their expression  or an increased half-life of cytokines due to delayed proteolysis resulting from binding of advanced glycation end products . Second, diabetic patients may produce higher levels of innate and type 1 cytokines because of higher bacterial load at the time of tuberculosis diagnosis, as observed in the mouse model of tuberculosis and chronic diabetes . In the early stages of tuberculosis infection in the mouse model (comparable to a period well before the patient consults the physician), mice with diabetes lagged in IFN-γ production. However, over time, mice with diabetes developed higher IFN-γ levels and bacterial loads in the lung, possibly because of higher antigenic stimulus. This pattern may be analogous to that in patients with chronic diabetes and chronic hyperglycemia who subsequently appear at a tuberculosis clinic. Unfortunately, our tool to assess bacterial load in patients is direct smear, which is insensitive and imprecise. In a previous retrospective study involving >5000 patients with tuberculosis, we showed that those with self-reported diabetes had a significantly higher frequency of positive smear results . With a limited number of cases available for prospective analysis, we have not yet seen a statistically significant difference in the proportion of diabetic patients with positive smear results (97% in the group of patients with diabetes and 90% in the group of patients without diabetes; table 1).
Interpretation of the meaning of up-regulated cytokine levels in diabetic patients with respect to tuberculosis must be performed with an open mind. First, our studies only evaluated the response to PPD, and immunity to M. tuberculosis in vivo is well known to be directed to complex structural and secreted products (e.g., lipoproteins, glycolipids, and mycolic acids) [38, 39]. Second, we selected patients with tuberculosis who did not have diabetes (or HIV infection) as control subjects, because they were the best group to compare with the case patients. However, we recognize that our control subjects comprise a heterogeneous group of individuals who are also “susceptible” to tuberculosis. Finally, we are aware that local organ and systemic immune responses may be related, but our observations of the peripheral response must be extrapolated with caution to the events in the primary site of infection, the lung.
Two previous studies involving patients with tuberculosis have reported associations between the cytokine responses to M. tuberculosis in diabetes [40, 41]. In contrast with our findings, both of these studies suggest lower levels of IFN-γ in response to M. tuberculosis antigens in diabetic patients. Closer examination of the data in these studies leads to possible explanations for the discrepancies. First, in the published studies [40, 41], the blood (or PBMCs) was diluted in cell culture media prior to lymphocyte stimulation. In our study, lymphocytes were stimulated in whole blood, preserving the blood glucose environment and other plasma factors (e.g., advanced-glycation end products). We then added costimulatory antibodies to boost memory T cell responses . Second, our study population was Hispanic, and genetic (ethnic) differences may also be important. Third, different mycobacterial antigens were used for stimulation. Fourth, there could be differences in the level of blood glucose control between study populations. Finally, we included patients who had received up to 8 days of treatment, which may have contributed to the discrepancies (although neither our univariate analysis nor our multivariate models were visibly affected by this variable). The lack of understanding of these apparent variations between studies illustrates the scant knowledge regarding the biological basis of the association between tuberculosis and diabetes.
In summary, we have developed an in vitro system to detect differences in the immune response to M. tuberculosis in diabetic patients and have determined that the most sensitive predictor of detectable differences is chronic hyperglycemia. These data provide the foundation for additional studies focused on specific pathways. The ultimate goal is to have a precise understanding of the cellular and molecular events underlying the association between tuberculosis and diabetes.
We thank Diana Gomez, Perla Martinez, and Dr. Kristy Murray, for technical support; Izelda Zarate, for field coordination; and Drs. Gonzalo Crespo and Ricardo Avila-Reyes, for logistical support at the Secretaría de Salud de Tamaulipas.
Financial support. National Institutes of Health (NIH P20 MD000170–04 and NIH 1U54RR023417–01), University of Texas Health Science Center at Houston, School of Public Health (PRIME-UTHSC-H), and Borderplex (UTHSCH-UTB-UT-PANAM).
Potential conflicts of interest. All authors: no conflicts.