Background
Approximately 36.9 million people worldwide were living with HIV infection in 2017, and 6.3 million new cases of tuberculosis (TB) were reported in 2016 [
1]. Of these 6.3 million people with TB, 7.6% (476,774) were also HIV-1 infected, of whom 85% were on combined antiretroviral therapy (cART), making TB the most common opportunistic infection leading to death among HIV-1 patients [
1]. Approximately 69,500 new cases of TB were reported in 2016 in Brazil, and 9.4% of these cases were associated with HIV-1 infection [
2]. In Rio de Janeiro, the Brazilian state where the individuals included in this study were recruited, the incidence rate of TB was 61.2/100,000 inhabitants in 2016, 8.9% of which also had an associated HIV-1 infection [
2].
Genetic studies have provided valuable insights into the resistance, susceptibility, and progression of infectious diseases since the enormous diversity of phenotypes associated with these diseases reflects the heterogeneous composition of host genotypes. Studies have suggested that both innate and adaptive immunity are involved in the pathogenesis of infectious diseases [
3,
4]. Therefore, the characterization of immune response genes is an important step in understanding the factors that can lead to TB and/or HIV-1, TB-HIV co-infected individuals, and IRIS onset. It is known that HLA (human leukocyte antigen) class I and KIR (killer-cell immunoglobulin-like receptor) genes influence the outcomes of HIV-1 infections and TB [
5‐
7]. In particular, the HLA-B locus plays a dominant role in the selection of cytotoxic T-lymphocyte (CTL) responses when compared with other class I molecules [
7‐
9]. HLA-C has a dual role of presenting antigens to CTL and serving as ligands to KIR receptors on NK cells, thus regulating the lysis of target cells mediated by NK cells [
10]. Many KIR genes and KIR-HLA-B/KIR-HLA-C pairs have been associated with distinct outcomes in the context of HIV-1 infection [
6,
11‐
14]. In the same way, genetic studies involving protection or susceptibility to pulmonary tuberculosis have also highlighted the role of HLA-B and KIR genes, as well as KIR + HLA-C pairs [
5,
15‐
18].
On the other hand, studies associating both innate and adaptive immune response genes with outcomes of TB-HIV co-infection are scarce. Some of the markers already described are HLA-A, −B, and -DRB1 alleles [
19,
20]. The management of TB-HIV co-infected individuals may have specific characteristics that can bring complexity to its dynamics. One example is the improvement of survival provided by the use of cART during TB treatment, which can restore immune function [
21]. However, simultaneous treatment with anti-TB drugs and cART can lead to a paradoxical clinical worsening with exacerbation of the immune response, known as immune reconstitution inflammatory syndrome (IRIS) [
21,
22]. The syndrome is currently classified into two forms, named paradoxical IRIS and unmasked IRIS. In paradoxical IRIS, the signs and symptoms of a pre-existing opportunistic infection, partially treated, recur, or worsen intensely despite a positive response before cART [
23,
24]. Unmasked IRIS is characterized by the discovery of a previously undiagnosed/latent infection. In this way, the signs and symptoms of opportunistic infection do not appear initially, appearing after the introduction of cART [
23,
24]. IRIS has been associated with a large variety of other pathogens and autoimmune diseases [
21,
25,
26], but mycobacterial infections are the most prevalent cause of IRIS [
21,
27,
28]. TB/HIV-IRIS occurs in 4–54% of patients starting cART during TB treatment [
29], depending on various features. In South America, this estimated incidence was 10% in an extensive meta-analysis comparing TB/HIV-IRIS cases from different parts of the world [
30]. The few Brazilian studies regarding IRIS report an estimated incidence of approximately 12%, which limits the recruitment and analysis of these individuals [
31,
32]. Pathogenic mechanisms involved in IRIS development have been suggested, such as antigenic load, degree of immune restoration after treatment with cART, and genetic susceptibility of the host, and there is evidence that these mechanisms can interact with each other and together cause the syndrome [
23,
25,
33]. However, studies linking host genetics to the pathogenesis of IRIS are still scarce [
34‐
37]. Based on the microarray analysis of gene expression of isolated monocytes, Tran et al. (2013,2014) [
34,
35] demonstrated upregulation of genes related to the role of pathogen pattern recognition to bacteria and viruses and the complement system, highlighting the potential role of monocytes and complement in the predisposition/development of TB-IRIS. Besides, Affandi et al. (2013) [
36] demonstrated that the susceptibility to TB-IRIS was associated with the presence of specific single nucleotide polymorphisms (SNPs) of cytokine-related genes.
Moreover, clinical risk factors already known to be associated with IRIS pathogenesis are (a) low baseline CD4
+ T-cell count (< 50–100 cells per mm
3) combined with a short time interval between the beginning of TB treatment and cART [
38‐
42] and (b) dissemination of TB to extrapulmonary sites, possibly reflecting a high bacterial load [
43,
44]. Nevertheless, despite the few biomarker descriptions associated with IRIS, there is still no one capable of predicting IRIS development currently being used in the clinical practice.
Innate and adaptive immunity are directly involved in the pathogenesis of IRIS [
3,
26,
44,
45]. Characterization of immune response genes is an important approach to assess genetic profiles that could be associated with susceptibility/resistance to the syndrome. In this way, this study aimed to investigate the distribution of HLA-B, HLA-C, and KIR genotypes and their potential influence on susceptibility and/or resistance to TB and/or HIV-1 as well as on the occurrence of TB-IRIS.
Discussion
The growing interest in the role of host genetic factors in the dynamics of infectious diseases is at least in part fueled by the possibility of finding predictive biomarkers of disease outcomes, such as the occurrence of IRIS in TB-HIV co-infected individuals, contributing to improving clinical management in an attempt to avoid severe disease complications. Several reports have associated polymorphic genes with infectious diseases in different populations and ethnic groups [
5,
7,
12,
60,
61]. Host genetic factors have been consistently linked to variations in both susceptibility and resistance to HIV-1 infection and TB [
7,
15,
62,
63].
Regarding TB-HIV co-infected individuals, there are few host factors associated with protection or susceptibility mechanisms. The immunological mechanisms underlying the development of IRIS are not yet clearly understood [
4,
37,
64,
65]. However, some authors have described potential biomarkers as predictors of IRIS development, for instance, interleukin-18 (IL-18) [
4], CXCL10, and IFN-α2 [
65]. Similarly, Conesa-Botella et al. reported that tumor necrosis factor (TNF), interferon-gamma (IFN-γ), IL-6, and IL-18 were significantly higher in patients with IRIS [
66]. Increased frequencies of IFN-γ-producing cells by Elispot in response to PPD and 38 kDa/CFP-10 antigens were also observed for IRIS patients in a previous study by our group [
46]. Concerning natural killer (NK) cells, Pean et al. showed that patients with IRIS had a higher proportion of NK cells degranulation levels of these cells were predictive markers of IRIS development among Cambodian TB-HIV co-infected individuals [
3]. Our group performed a similar analysis for a subset of patients here included, but no difference was observed in NK degranulation between IRIS and non-IRIS groups [
47]. Also, other groups reported elevated frequencies of KIR-γδ T-cells [
67] and CD69+ NK cells [
68] in TB-IRIS patients during pre-ART, suggesting that these cells may play a role in IRIS-associated pathology. However, it is not completely elucidated which of these potential biomarkers might have clinical application in predicting IRIS.
In the present study, we examined the distribution of HLA-B, HLA-C, and KIR genes in TB and/or HIV-infected patients and investigated the putative role of these genes in the occurrence of TB/HIV-IRIS. The individuals included in the present study had their HLA-B, HLA-C, and KIR genes determined, and the frequency data observed corroborated what has been described for the HIV-1-infected population [
12,
69] and the general Brazilian population [
70‐
75]. HLA-B*08 and female gender were associated with protection against TB onset in the studied population. On the other hand, the KIR2DS2 gene was associated with an increased risk for TB onset (Table
2). To the best of our knowledge, HLA-B*08 has not yet been associated with TB protection. However, an increased frequency of this allele was described among TB-HIV coinfected individuals and HIV-1 infected patients with rapid disease progression, reflecting different roles for this allele in the context of TB and HIV-1 infection [
76‐
79]. Many studies have established links between sex-specific factors and the differential susceptibility or protection to some infectious diseases [
80,
81]. TB rates are significantly higher in men than in women [
1]. Herzmann and collaborators observed a higher frequency of active TB among men, which could lead to an increased risk for disease progression [
82]. KIR2DS2 recognizes HLA-C molecules of the C1 group [
83]; there is no previous report associating KIR2DS2 with TB susceptibility. Instead, KIR2DS2 has been associated with rapid disease progression and robust immune activation, accelerating the progress to AIDS [
84,
85], and promoting a higher risk to acute lymphoblastic leukemia [
86].
Not carrying KIR2DL3 and the carriage of HLA-C*07 were protective factors for TB onset among HIV-1-infected individuals studied here (Table
2). KIR2DL3 recognizes HLA-C molecules of the C1 group [
83]. Previous studies have shown that different levels of susceptibility to
M. tuberculosis may be due to variations in KIR receptors and, consequently, in the repertoire of NK cells [
87‐
89]. In the context of TB, a higher prevalence of KIR2DL3 among TB patients has been observed in several studies [
15,
18,
90,
91]. Biberg-Salum et al. [
92] showed that HLA-C∗07 allele conferred protection against the development of cytomegalovirus retinitis in Brazilian AIDS patients.
It is noteworthy that all patients who developed TB/HIV-IRIS in our analyses were males. The predominance of males among IRIS patients had already been documented in other studies, but in most of them, there was no association with increased risk of IRIS onset [
4,
38,
93]. However, an increased risk of being diagnosed with IRIS was reported for men [
93]. We could not confirm this association, given the lack of women with IRIS in our study, which prevented the inclusion of the gender variable in the statistical models.
Interestingly, an increased risk for IRIS onset among TB-HIV co-infected individuals was found among those having a CD8 count ≤500 cells/mm
3; carrying the KIR2DS2, the HLA-B*41, and the KIR2DS1 + HLA-C2 pair; as well as not carrying KIR2DL3 + HLA-C1/C2 and KIR2DL1 + HLA-C1/C2 pairs (Table
3). HLA-B*41 allotypes have already been associated with susceptibility to TB in patients with AIDS from the northeast region of the state of São Paulo [
20], but no association with IRIS has been described for this allele yet. The frequency of the HLA-B*41 allele is low in different populations (Allele Frequency Net Database), differing from the frequency found in the IRIS cases included in the present study. The KIR2DS2 gene was also associated with IRIS onset among TB-HIV co-infected individuals in the present study. The high frequency of this gene described across all studied groups (51.2%) was similar to those observed in several other populations, such as on the African continent (> 54%) and in the Cambodian population (49.9%) [
90], where the occurrence of IRIS is higher than that observed in this study [
31].
The results regarding activating KIR receptors (KIR2DS2, KIR2DS1 + HLA-C2, and KIR2DS5) together with the lack of inhibitory KIR receptors (KIR2DL3 + HLA-C1/C2 and KIR2DL1 + HLA-C1/C2) might reflect a high functionality of NK cells, suggesting that the presence of these activating genes modulates the NK cell response. This mechanism may be either by no recognition of the activating genes of the infected cells, due to lack of ligands in the target cell, or due to overriding of the activation signal by the inhibitory signal delivered to NK cells when both activating and inhibitory genes bind to their ligand on the surface of the target cell [
94‐
96]. Therefore, this might lead to an escape from the infected cells, resulting in the exacerbation of the pathogenesis of IRIS or HIV-1 infection and TB itself. Future studies should address the functional characterization of these genes and their respective HLA ligands.
To the best of our knowledge, this is the first study showing the scenario of HLA-B, HLA-C, and KIR gene frequencies in a population of HIV-1-infected patients with TB. Importantly, the frequencies of these genes between individuals with and without IRIS were also determined. Our results suggest the participation of the clinical and genetic markers, which were associated with the related TB-HIV outcomes in the immunopathogenic mechanisms related to the conditions studied here. It is relevant to point out that some limitations of the current study should be noted, mainly concerning the limited sample size and the low frequency of TB/HIV-IRIS cases. Therefore, additional studies with larger populations and suitable power analyses might be helpful to a better understanding of the importance and role of genetic host markers in the context of TB and/or TB/HIV-IRIS.
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