Cell-mediated immunity plays an important role against all infectious diseases. Production of cytokines and chemokines is an integral component of immune response. Cytokine storm has suggested to be an important factor responsible for the development of symptoms in infected patients.[1,2] The literature has shown the elevated levels of pro- and anti-inflammatory cytokines in the acute phase of scrub typhus (ST) illness. Early host inflammatory responses seem to play a key role in determining the fate of the host infected with Orientia tsutsugamushi.[4,5] The presence of both pro- and anti-inflammatory cytokines during acute ST infection is an example of the infected host trying to maintain homeostasis during infection.
Experimental studies in mice models have suggested that protective immunity to ST is mediated by T-helper-1 (Th) cells, as demonstrated by raised interferon-gamma (IFN-g)-producing T-cell. It is well known that Th1 immune responses are crucial to protect against the intracellular pathogens. However, the inclination toward Th2 lineage such as elevated interleukin (IL)-6 and IL-10 has also been variably observed in ST. Thus, there seems a concurrent expression of CD4+ Th-1 and Th-2 cytokines in ST patients.
A little attention has been given to the chemokine and cytokine responses in the immunopathogenesis of ST. The immune mechanisms that regulate the degree and quality of the cellular invasion to the site of the infection need to be explored. Some literature is available on the role of different cytokines in disease processes.[3,8] However, the in vivo studies on human infections are lacking.
Due to this paucity of research in this domain, the current study aimed to analyze the concentrations of IL-2, IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, transforming growth factor (TGF)-β, and human macrophage inflammatory protein (HMIP)-1 β in the sera of ST-positive patients during the acute phase of illness.
Materials and Methods
This is a prospective cross-sectional study conducted for 2 years. The study group comprised 112 ST patients presenting with acute febrile illness with or without acute respiratory failure and acute renal failure. Clinical signs and symptoms were recorded on a predesigned pro forma. ST infection was confirmed by detecting immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA) test. An informed consent was taken from these patients before their inclusion in the study. This study has been approved by the Institutional Ethical Committee of the hospital, and clearance has been issued on June 24, 2015.
Thirty blood samples of healthy blood donors from the blood bank were randomly chosen as controls.
Collection of specimens for diagnosis and cytokine analysis
The blood was aseptically collected in sterile plain vials from all patients for serology. Serum was separated by centrifugation at 3000 rpm for 15 min and stored at −20°C till further testing. Sera collected from acute febrile illness patients were evaluated to detect ST by IgM ELISA and IFA IgM.
Immunoglobulin M enzyme-linked immunosorbent assay
All sera were tested for the presence of IgM antibodies against 56 KDa antigen of ST by IgM ELISA (InBios International Inc. Seattle, WA, USA). The results for ST IgM ELISA were interpreted according to the manufacturer’s instructions.
Indirect immunofluorescence assay
The IFA kits were procured from Fuller laboratories, California, USA. IFA was performed to detect IgM antibodies against O. tsutsugamushi. The IFA slides in kit had four strains: Boryong, Gilliam, Karp, and Kato. As per the manufacturer’s instructions, the slides were examined at 400× magnification. Samples were regarded positive when fluorescent, short, and pleomorphic rod forms appeared in any of the four antigen areas.
The serum cytokine levels in 112 ST-positive patients during the acute phase of illness were estimated by ELISA (Pro-inflammatory cytokines: IL-2, IL-6, and TNF-α ELISA Diaclone, France; Anti-inflammatory cytokines: IL-10 ELISA Diaclone, France, TGF-β2 ELISA Qayee Bio, and HMIP1-β ELISA Qayee Bio). The serum level of all cytokines was also evaluated in 30 healthy controls (HC) chosen from the blood bank. A standard curve of optical densities versus concentrations of different cytokines was generated to determine their concentrations in serum samples.
Statistical analysis was performed with the IBM SPSS Statistics 20 (IBM Inc. Armonk, New York, USA) and MedCalc 16.1 Software. The evaluation of differences in cytokine levels between two groups was performed using nonparametric Mann–Whitney U-test. For descriptive statistics, the mean and interquartile range are reported. The Spearman’s rank correlation test was used for the assessment of correlation. P < 0.05 was considered statistically significant. MedCalc 16.1 used receiver operating characteristic (ROC) curve to assess the diagnostic value, sensitivity, and specificity of cytokines in ST. The comparisons of area under curve (AUCs) were performed using z-test. The optimal diagnosis threshold was determined according to the Youden index J, and relative sensitivity and specificity were calculated.
All 112 cases included in the study were confirmed to be ST-positive by IgM ELISA and IFA. The median age of the patients was 47 ± 14 years (range 18–74 years), and the male-to-female ratio was 1.11 (59/53).
The clinical profile of 112 ST patients with acute febrile illness: chills 91 (81.2%), myalgia 72 (63.2%), headache 70 (62.5%), cough 24 (41.3%), tachypnea/respiratory distress 49 (43.7%), yellow urine 60 (53.5%), reduction in urine output 21 (18.7%), lymphadenopathy 68 (60.7%), and pain in abdomen 7 (12%), whereas rashes were present in 2 (1.8%) cases. Chills and body ache were the most common manifestations in the majority of cases, whereas severe cases mainly presented with respiratory system involvement in the form of breathlessness and respiratory distress [Figure 1].
The mean and median values of IL-2, IL-6, IL-10, TNF-α, TGF-β, and macrophage inflammatory protein-1 (MIP-1) β among ST patients and HC were calculated, and the means were compared using the Mann–Whitney U-test and independent t-test [Table 1]. The mean level of the cytokines (IL-6, IL-10, TNF-α, and HMIP1-β) was significantly elevated in the ST patients compared to the control group (P < 0.0001) [Table 1 and Figure 2]. However, the mean of IL-2 and TGF-β did not differ significantly between ST patients and HC (P = 0.0653, P = 0.0386, respectively). The mean and median values of IL-2 in ST patients and HC were found to be 64.38 (61.679–67.087), 63.89 (59.680–67.104), 66.80 (65.004–68.593), and 67.25 (63.890–68.638), respectively. Box plot analysis showed that most of the IL-2 values were comparable among two groups with majority clustering between 60 and 70 pg/ml (74.81–55.48 [HC] vs. 95.83–47.91 [ST]), which were not significant (P = 0.0653); whereas, the IL-6 and IL-10 values among ST patients were found largely scattered, with majority of the values above the 75th percentile of HC (IL-6 [14.64–5.45 (HC) vs. 348.1–1.6 (ST), P < 0.001]; IL-10 [15.66–7.29 vs. 560.53–1.01, P < 0.001], respectively). There were wide gaps between the highest and lowest values of IL-6 and IL-10 with no specific clustering. TNF-α levels among ST patients were mainly concentrated around 60–80 pg/ml with a few values near 300 pg/ml, whereas HC values were mainly concentrated between 50 and 60 pg/ml (mean and median values of ST patients and HC 187.05 [78.387–295.71] and 72.90 [63.750–80.117] and 56.62 [51.802–60.375] and 56.81 [49.948–62.007], respectively). Broad scattering of TGF-β values in ST patients and HC (454.66–12.38 vs. 14.99–254.57) was observed with no significant difference between two groups (P = 0.0386, Mann–Whitney U-test U: 635.0). HMIP-1 β levels were found low in both the groups compared to other cytokine levels, but there was a significant difference between ST patient levels and HC (72.45–1.81 vs. 9.88–1.81, P < 0.001, Mann–Whitney U-test: 307.50).
On analysis of ROC, a biomarker with AUC = 1 or near discriminates individuals perfectly as diseased or healthy. The AUCs for IL-6, IL-10, TNF-α, and HMIP-β were 0.887 (95% confidence interval [CI] 0.802–0.945), 0.851 (95% CI 0.760–0.918), 0.745 (95% CI 0.641–0.832), and 0.823 (95% CI 0.727–0.896), respectively, indicating that IL-6, IL-10, TNF-α, and HMIP-1 β were effective biomarkers for ST [Figure 3]. The significant cutoff serum levels for IL-2 was 64.81 pg/ml (67.35% sensitivity and 70% specificity), IL-6 was 7.95 pg/ml (84.9% sensitivity and 86.7% specificity), IL-10 was 8.84 pg/ml (86.2% sensitivity and 80.0% specificity), TNF-α was 66.27 pg/ml (56.9% sensitivity and 80.0% specificity), TGF-β was 110.91 pg/ml (37.9% sensitivity and 96.7% specificity), and HMIP-1 β was 9.72 pg/ml (63.7% sensitivity and 96.67% specificity) [Table 2]. Around 90.1% (101/112) of patients displayed abnormally high levels of IL-6. IL-10, TNF-α, and HMIP-1 β levels were raised in 97 (86.6%), 63 (56.2%), and 69 (61.6%) ST patients, respectively. The increased TNF-α levels were mainly found in ST patients with severe sign and symptoms in the form of tachypnea/breathlessness, whereas only 26 (23.2%) showed raised levels of IL-2. TGF-β levels were also found to be elevated in few patients, 42 (37.5%).
Immunological profile and cytokine analysis is the least scrutinized aspect in ST disease. A very few studies have been done in India, especially in the northern part of the country. In this study, we explored the level of different cytokines during the acute phase of illness in ST-positive patients.
The clinical profile of our study group showed usual signs and symptoms in the form of lymphadenopathy 68 (60.7%), chills 91 (81.2%), and myalgia 72 (63.2%). Severe cases mainly presented with respiratory involvement, 49 (43.7%), in the form of tachypnea/breathlessness. Fortunately, none of the patient developed severe complication in the form of coma, severe jaundice, intubation, etc., requiring prolonged hospital care. All the patients fully responded to treatment with zero mortality. However, severe cases were associated with altered cytokine profile.
In our study, the levels of IL-6, IL-10, TNF-alpha, and HMIP-1 were found significantly raised during the acute phase compared to HC (P < 0.0001). However, there was no considerable difference in the levels of IL-2 and TGF-beta between the ST patients versus HC group. They were found to be less discriminatory between healthy and diseased (AUC 0.710, 95% CI 0.597–0.806 and 0.623, 95% CI 0.529–0.723, respectively).
Increased chemokine and cytokine response have proven an association with increased susceptibility to ST in animal models.[8,9] Various studies on IL-10, TNF-α, and other cytokines in humans demonstrated their role during the acute phase of ST infection.[2,3,10] Significantly higher levels of IL-6, IL-10, and TNF-α with other cytokines have been reported in earlier studies.[2,3,10,11] Our findings are in concordance to these reports.
IL-6 is a cytokine with both pro- and anti-inflammatory properties. Its role has been observed in various diseases such as atherosclerosis, Alzheimer’s, autoimmune disease, different cancer, and recently in COVID 19. Recent reports showed the increased levels of IL-6 in ST patients suggesting its stronger anti-inflammatory activity.[2,11] The elevated IL-6 levels demonstrate Th-2 cell response, which in turn leads to B-cell differentiation. Around 101 (90%) patients demonstrated raised IL-6 levels in our study. Recent reports on COVID-19 revealed its association in severe disease progression. One similar report proposed its association with development of hypoxia in COVID-19 cases. As respiratory system involvement was the most common presentation representing the severity of illness in our cases, the raised IL-6 levels may have some association with such kind of presentation.
The anti-inflammatory and Th1 cytokine assessed in this study were IL-2 and TNF-α, however, signatory cytokine for Th-1 cells is IFN-γ. In our previous report, the levels of IFN-g in ST patients were comparable to healthy adults. The current report revealed the comparable levels of IL-2 (mean ST: 64.38, 95% CI 61.67–67.08 vs. mean HC 66.80, 95% CI 65.00–68.59) with only 26 (23.2%) patients showing raised levels. TNF-α levels were found to be high in 63 (56.2%) cases with few values as high as 298.77 pg/ml. These high levels of TNF-α showed relationship with the severity of ST disease as significantly higher values were found in patients with breathlessness/tachypnea. The literature is available on mixed role (protective vs. fatal role) of TNF-α in the pathogenesis of various infectious diseases.[3,16–18] During the acute phase, TNF-α levels might predict the severity of ST disease; however, its association with disease progression or as host defense is yet to be proven in humans. On the basis of the current findings, we can only conclude that TNF-α is an essential component of host defense mechanism; its underproduction may lead to the proliferation of the pathogen, whereas overproduction of TNF-a may be detrimental to the host.
TNF-α is a decisive proinflammatory cytokine in the host’s response to infection, whereas IL-10 apart from being an anti-inflammatory cytokine, is also a potent inhibitor of monocyte/macrophage activation and TNF-α production. IL-10 ensures homeostasis by regulating the immune response. It has been reported that an imbalance of these two cytokines, with low levels of TNF-α and high levels of IL-10, has been associated with a better prognosis in other bacterial infections.[16,19] In this study, 97 (86.6%) patients showed high serum levels of IL-10 in the acute phase. Kramme et al., 2009, documented raised levels in 50% of patients. He also demonstrated a significant association between IL-10 levels with low levels of TNF-α. Studies on mice and murine models documented that increased levels of IL-10 downregulate TNF-a levels.[8,20] Our findings in humans also match these observations. Although the diverse mechanism has been proposed by which IL-10 inhibits TNF-a production in other diseases, a clear relationship between TNF-α and IL-10 in ST remains unclear. However, our study confers the previous findings, but more extensive study is required to understand the mechanism of inter-regulation of TNF-α and IL-10.
Not much work has been done on IL-2 and TGF-β association in ST in human cases. In our study, the level of these cytokines apparently seems not to be associated in immune regulation of disease. However, their role needs to be explored in severe ST cases. With comparable IL-2 levels and moderately raised levels of TNF-α in majority of cases, we conclude that IL-10 may have a role in the downregulation of Th-1 cells cytokines.
Protective immunity against O. tsutsugamushi is mainly due to the activation of macrophages leading to cytokines, chemokines, and T-cell response.[4,5] MIP-1 β chemokines are secreted by macrophages, which in turn has a role in the activation of Th-1 cell proliferation. The increased levels of MIP-1 β in our study are indicating its role in ST disease and Th-1 activation which can be demonstrated through the levels of TNF-α. A study on mice model found the higher levels of MIP-1 β and TGF-β after ST infection. Astrup et al. also documented the greater levels of MIP-1 β in their ST patients.
Cytokines involved in immune regulation should be maintained at appropriate concentrations with proper antimicrobiological chemotherapy. Studies have shown symptomatic improvement in ST patients on treatment with tetracyclines, which could be due to the potential of tetracyclines to decrease the overproduction of cytokines. This may also be the possible reason that our patients responded without lending into severe disease. Another reason for such clinical presentation may be the circulating strain which could be less immunogenic and less invasive. However, this aspect needs to be explored.
IL-6, IL-10, TNF-α, and HMIP-1 β levels were found to be increased in ST patients during the acute phase. The elevated IL-6 levels may be related to the development of respiratory complications. TNF-α levels in the acute phase may predict the severity of ST disease. The protective and downregulatory role of IL-10 may also be linked to favorable outcomes and less severity of ST patients. Timely suspicion and treatment with tetracyclines may lead to effective management of disease.
In this study, the cytokine profile revealed a proper balance between levels of different pro-inflammatory cytokines, chemokines, and immune-regulatory cytokines. This could be one of the reasons of clinical presentation with mild-to-moderate disease severity.
Financial support and sponsorship
This work was funded by grants from SERB, the Department of Science and Technology (DST), India, under the Early Career Research Advancement Scheme.
Conflicts of interest
There are no conflicts of interest.
The authors are thankful to SERB, DST, for providing the funds, and Mr. Sanjay Sharma, Department of Microbiology, for helping with the technical Work.
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