Immunological Profile of HIV-Infected Patients with Tuberculosis Associated- Immune Reconstitution Inflammatory Syndrome: A Systematic Review

Objective: This study systematically reviews the literature that describes the immunological profile associated with the development of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) in HIVinfected individuals. Methods: Between the primary and secondary searches, a total of 20 articles were selected for the final analysis. Results: The results obtained herein indicated that TB-IRIS was associated with the recovery of Mtb-specific immune response, demonstrated by an increased frequency of specific IFN-g-producing cells and specific multifunctional T-lymphocytes (TNF and IFN-γ-producing). In addition, an increased production of inflammatory cytokines and chemokines was found in TB-IRIS patients compared to non-IRIS individuals. Conclusion: These data suggest that expansion of Mtb-specific cells may not be the main factor for the occurrence of IRIS. Further studies are needed to better evaluate the dynamic of restoration of Mtb-specific memory cells and to clarify the role of innate immune responses in immunopathogenesis of TB-IRIS patients.


Introduction
Highly active antiretroviral therapy (HAART) had a major impact in reducing mortality and morbidity associated with AIDS and a significant improvement in patients' quality of life [1,2]. Although HAART is effective at controlling viral replication and inducing partial restoration of CD4 + T-lymphocyte repertoires, around 16% of treated patients experience a clinical deterioration [3,4]. They present an overwhelming inflammatory response against pre-existing antigens that is named inflammatory immune reconstitution syndrome (IRIS) [5,6]. This syndrome results from the immune system's restored ability to mount a potent inflammatory response after HAART. IRIS can manifest as a paradoxical response, in which clinical worsening occurs when patients start on pathogen-specific therapy and HAART simultaneously. Alternatively, it can occur as an unmasking IRIS, in which a latent opportunistic infection is identified following HAART initiation [7,8].
The immune reconstitution following HAART is characterized by an increase in the number of CD4 + T-lymphocytes, restoration of lymph oproliferative response to memory antigens, and a shift from a T helper (Th) type 2 to a type 1 cytokine profile, with an increase in IL-2 and IFN-g levels [14][15][16][17]. Although the immunological mechanism involved in TB-IRIS remains partially unclear, it has been suggested that the intense inflammatory response results from an exaggerated antigen-specific response [18,19]. Moreover, the production of spontaneous pro-inflammatory cytokines and chemokines [19] and/or an imbalance in the immune regulatory response [20] are found in the course of TB-IRIS. Studies assessing the immune pathogenesis of TB-IRIS are scarce, include small samples of patients and describe different aspects of the immune response. The present study aims to systematically review the pertinent literature to describe the immunological profile associated with the development of TB-IRIS in HIV-infected individuals.
Studies were selected according to the following criteria: original articles and articles whose results presented the assessment of markers of the Mtb-specific immune response in HIV-infected patients with TB-IRIS. The exclusion criteria were: letters to the journals and animal model studies. These criteria were first applied to titles and abstracts and then to full text articles. The final article selection was defined by consensus between the two researchers. Secondary search was additionally performed from references included in the original articles.
The following information was systematically extracted from each article: (1) basic information (title, year, authors, objectives, and keywords), (2) study design, (3) methods used for evaluation of the immune system (innate and antigen-specific response), (4) subjects (setting, sample, data collection, procedures and tools), and (5) results obtained. The systematic literature review was structured according to the PRISMA checklist.
CD4 + T-cell count, HIV viral load and the number of Mtb-specific IFN-g-producing cells evaluated by enzyme-linked immunospot assay (ELISPOT) of TB-IRIS patients and non-IRIS were extracted from all articles where information was available. The data of TB-IRIS patients and non-IRIS were compared using U Mann-Whitney test (p<0.05).

Discussion
The results of this systematic review show that the presence of TB-IRIS was concomitant with the restoration of the Mtb-specific immune response. In the majority of the evaluated studies (11/15), a higher number of Mtb specific-IFN-γ producing cells [18,20,22,35] and of specific multifunctional T-lymphocytes (IFN-γ and TNF-producing) [22,28] as well as higher production of Th1 cytokines [18,23,35,36] were found in TB-IRIS patients compared to non-IRIS individuals. However, the peak in the number of antigen-specific IFN-γ-producing cells did not always coincide with the onset of IRIS, occasionally occurring after IRIS resolution [22,25]. In only four out of 15 studies, three using IGRA and one using ELISPOT, differences in IFN-γ production in response to Mtb antigens were not reported between groups [29,31,33]. Interestingly, the aforementioned study using ELISPOT also found a low production of IFN-γ in response to cytomegalovirus and influenza antigens in TB-IRIS patients, suggesting that those patients were immunosuppressed [29]. None of studies using IGRA found any difference in the production of IFN-g between TB-IRIS and non-IRIS groups. This could indicate that ELISPOT sensitivity to Mtb antigens may be higher than IGRA in patients with advanced HIV infection [38,39]. specific IFN-g-producing cells of TB-IRIS patients and non-IRIS individuals using ELISPOT. Data were extracted as median from all articles that used ELISPOT to quantify IFN-g-producing cells in response to Mtb antigens (PPD, ESAT-6, 85B, 38KD, Acr 1 and 2 and lipomannan) [18,20,22,30,35]. TB-IRIS group: median: 3,216 SFC/106 PBMC, IQR 1,528-4,616 SFC/106 PBMC; non-IRIS group: (441.5 SFC/106 PBMC, IQR 233.5-563.3 SFC/106 PBMC). Mann-Whitney test (p<0.05).

Immunological assay Main results Reference
Plasma cytokines (ELISA) Higher IL-6 level in TB-IRIS patients compared to non-IRIS individuals [37] Cell phenotyping (WB, flow cytometry) Increased TNF, IL-6, IL-1β, IL-10, RANTES, and MCP-1 levels and high frequency of activated CD4 + T-cells in TB-IRIS patients [18] Cell phenotyping (WB, flow cytometry) Similar frequencies of activated CD4 + and CD8 + T-cells and CD4 + Treg cells among TB-IRIS and non-IRIS individuals [20] Cell phenotyping (PBMC, flow cytometry) Higher frequency of activated CD4 + T-cells and CD4 + Treg cells (CD25 + CD127 low and CTLA-4 + ) in TB-IRIS patients compared to healthy individuals. [30] Cell phenotyping (WB, flow cytometry) Lower frequency of TCRγδ and Vδ2 + T cells expressing CD94/ NKG2 and CD158ah, b in TB-IRIS patients compared to non-IRIS individuals. [22] Plasma cytokines (flow cytometry and ELISA) Increased IL-18 and CXCL10 levels and decreased CCL2 in TB-IRIS patients compared to non-IRIS individuals. [19] Plasma cytokines (ELISA) At baseline, higher TNF and IL-10 levels in TB-IRIS patient compared to non-IRIS individuals. Higher IFN-γ levels in TB-IRIS patients after HAART compared to baseline. [34] Plasma cytokines (Luminex technology) Higher frequency of activated NK cell and C-reactive protein, IL-8, EGF, and HGF levels in TB-IRIS patients compared to non-IRIS patients. [24] Cell phenotyping (whole blood, flow cytometry) Higher expression of TLR2 on mDC and monocytes of TB-IRIS patients compared to non-IRIS individuals [35] Citation: Gois quantity of IL-2, IL-5, IL10, IL-12p40, IL-13, IL-15, IL-17A, TGF-β, and TNF transcripts in TB-IRIS patients compared to non-IRIS individuals [23] Cell phenotyping (WB, flow cytometry) Expansion of central memory CD4 + T-cells following HAART. [25] Cytokines levels (CSF, flow cytometry) Higher TNF, IFN-γ and IL-6 levels in TB-IRIS patients compared to non-IRIS individuals [27]  The low IGRA sensitivity observed could also be explained by the antigenic components of this test, ESAT-6, CFP-10 and TB 7.7, which are derived from region of difference 1 (RD1) in the Mtb genome. In fact, two studies that evaluated ESAT-6 response in patients with paradoxal TB-IRIS using ELISPOT detected low number of spot forming cells (SFC), whereas the number of SFC in response to protein purified derivate to Mtb (PPD) was high [18,30]. Conversely, higher IFN-γ-production in response to both PPD and RD1 antigens, including ESAT-6, was observed in patients with unmasking TB-IRIS compared to non-IRIS individuals [31]. As RD1 antigens are solely derived from Mtb, as opposed to PPD, it has been proposed that the inflammatory response in unmasking TB-IRIS would be triggered by antigens from live bacteria, while in paradoxal TB-IRIS that response is mainly triggered by antigens from dead bacteria [30]. Thus, IGRA could be useful distinguishing unmasking and paradoxal TB-IRIS.
This systemic review also found a low frequency of polyfunctional (IFN-g + IL-2 + TNF + ) CD4 + and CD8 + T-lymphocytes in response to PPD in TB-IRIS patients. The majority of patients had a specific multifunctional T-lymphocytes secreting IFN-γ and TNF, but not IL-2 in response to Mtb antigens stimulation [22,28]. These findings suggest that the quality of the specific immune response to Mtb antigens recovery is limited [28]. Mono-functional T-lymphocytes (CD4 + IFN-g + ) response is mainly found during persistent infection with high antigen load, as occurs during an infection associated with IRIS. Maintaining a high level of antigens impairs the establishment of a polyfunctional response capable of sustaining its own expansion and effector activity [40].
In addition to a high secretion of Th1 cytokines (IFN-γ, IL-2, IL-12) in response to Mtb antigens, several studies found higher production of cytokines and chemokines released from innate immune cells (TNF,  IL-6, IL-1β, IL-10, IL-18, CCL-5, CCL-2, CXCL10) [18,19,23,24,34,35,37] in patients with TB-IRIS compared to non-IRIS individuals. Non-specific release of proinflammatory cytokines and chemokines may induce the systemic inflammatory reaction present in IRIS. It has been proposed that once HAART controls the viral load and promotes the reconstitution of T-lymphocyte repertoires, specific lymphocytes could produce cytokines that stimulate macrophages which were previously infected by intracellular pathogens during the period of immunosuppression (AIDS). In turn, these macrophages and other cells of the innate immune system would secrete high levels of proinflammatory cytokines and chemokines, which would result in the inflammatory manifestations of IRIS [41].
Moreover, inflammatory response during TB-IRIS could also be caused by a dysfunction of regulatory T-lymphocytes. Two studies found similar frequencies of Foxp3 + CD4 + T-cells in patients with TB-IRIS and non-IRIS individuals [20,42]. Tan et al studying three patients with TB-IRIS, observed an increase in the proportion of Tcells with regulatory profile (CD4 + CD25 + CD127 low and CD4 + CTLA-4 + ) in comparison to healthy controls, but these findings have not been compared to patients who did not develop IRIS [30]. This review was unable to find studies evaluating the regulatory T-cell function in TB-IRIS patients. This review has some limitation we did not assess the evidence strength of results presented in the articles included and also the risk of bias in these articles.
In conclusion, the findings presented in this review suggest that during TB-IRIS an increase in the specific response to Mtb antigens occurs, as evidenced by a higher number of IFN-γ producing cells and by higher levels of cytokines. The potential role of innate immune response, with increased production of proinflammatory cytokines and chemokines, as well as activated NK cells and macrophages was also observed during TB-IRIS. Taken together, these data suggest that expansion of Mtb specific cells may not be the determining factor for the occurrence of IRIS. Further studies are needed to better evaluate the dynamic of restoration of Mtb-specific memory cells and to clarify the role of innate immune responses in immunopathogenesis of TB-IRIS. Modulating the proinflammatory cytokine storm observed during IRIS may be beneficial to patients by decreasing morbidity and mortality of TB-IRIS patients.