Clinical and genetic markers associated with tuberculosis, HIV-1 infection, and TB/HIV-immune reconstitution inflammatory syndrome outcomes.

BACKGROUND: Tuberculosis (TB) and AIDS are the leading causes of infectious disease death worldwide. In some TB-HIV co-infected individuals treated for both diseases simultaneously, a pathological inflammatory reaction termed immune reconstitution inflammatory syndrome (IRIS) may occur. The risk factors for IRIS are not fully defined. We investigated the association of HLA-B, HLA-C, and KIR genotypes with TB, HIV-1 infection, and IRIS onset. METHODS: Patients were divided into four groups: Group 1- TB+/HIV+ (n = 88; 11 of them with IRIS), Group 2- HIV+ (n = 24), Group 3- TB+ (n = 24) and Group 4- healthy volunteers (n = 26). Patients were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. The HLA-B and HLA-C loci were typed using SBT, NGS, and KIR genes by PCR-SSP. Unconditional logistic regression models were performed for Protection/risk estimation. RESULTS: Among the individuals with TB as the outcome, KIR2DS2 was associated with increased risk for TB onset (aOR = 2.39, P = 0.04), whereas HLA-B*08 and female gender were associated with protection against TB onset (aOR = 0.23, P = 0.03, and aOR = 0.33, P = 0.01, respectively). Not carrying KIR2DL3 (aOR = 0.18, P = 0.03) and carrying HLA-C*07 (aOR = 0.32, P = 0.04) were associated with protection against TB onset among HIV-infected patients. An increased risk for IRIS onset was associated with having a CD8 count ≤500 cells/mm3 (aOR = 18.23, P = 0.016); carrying the KIR2DS2 gene (aOR = 27.22, P = 0.032), the HLA-B*41 allele (aOR = 68.84, P = 0.033), the KIR2DS1 + HLA-C2 pair (aOR = 28.58, P = 0.024); and not carrying the KIR2DL3 + HLA-C1/C2 pair (aOR = 43.04, P = 0.034), and the KIR2DL1 + HLA-C1/C2 pair (aOR = 43.04, P = 0.034), CONCLUSIONS: These results suggest the participation of these genes in the immunopathogenic mechanisms related to the conditions studied. This is the first study demonstrating an association of HLA-B*41, KIR2DS2, and KIR + HLA-C pairs with IRIS onset among TB-HIV co-infected individuals.

Zinc finger and interferon-stimulated genes play a vital role in TB-IRIS following HAART in AIDS.

AIM: Co-infection in HIV-1 patients with Mycobacterium tuberculosis poses considerable risk of developing the immune reconstitution inflammatory syndrome (IRIS), especially upon the initiation of antiretroviral therapy (ART). Methodology & results: For transcriptomic analysis, peripheral blood mononuclear cells' whole gene expression was used from three patient groups: HIV+ (H), HIV-TB+ (HT), HIV-TB+ with IRIS (HTI). Pathway enrichment and functional analysis was performed before and after highly active ART. Genes in the interferon-stimulating and ZNF families maintained tight functional interaction and tilted the balance in favor of TB-IRIS. DISCUSSION & CONCLUSION: The functional impairment of interaction between ZNF genes and interferon-stimulated genes, along with higher expression of S100A8/S100A9 genes possibly forms the genomic basis of TB-IRIS in a subset of HIV patients while on highly active ART.

Nuclear Factor of Activated T Cells and Cytokines Gene Expression of the T Cells in AIDS Patients with Immune Reconstitution Inflammatory Syndrome during Highly Active Antiretroviral Therapy.

Background. The etiology of immune reconstitution inflammatory syndrome (IRIS) in AIDS patients after the initiation of HAART remains unknown. Several researches indicated that the development of IRIS is associated with the production and variation of cytokines, whose gene expression are closely related to the Ca2+/CN-nuclear factor of activated T cells (NFAT) pathway. Methods. We studied the expression of NFAT isoforms and their major target cytokines genes in peripheral blood CD3+ T cells of subjects through fluorescence quantitative PCR and explored the expression changes of these genes before and after HAART. Results. After the initiation of HARRT, NFAT1, IL-6, and IL-8 gene expression showed a reversal trend in the CD3+ T cells of the IRIS group and changed from low expression before HARRT to high expression after HARRT. In particular, the relative gene expression of NFAT1 was markedly higher compared with the other three isoforms. The IRIS group also showed higher NFAT4, NFAT2, NFAT1, IL-1ß, IL-10, IL-2, IL-18, and TNF-α gene expression than the non-IRIS group. Conclusion. This study suggested that high expression levels of IL-2, IL-6, IL-8, TNF-α, IL-1ß, IL-10, IL-12, and IL-18 can predict the risk of IRIS. The increased expression of NFAT1 and NFAT4 may promote the expression of cytokines, such as IL-6, IL-8, and TNF-α, which may promote the occurrence of IRIS.

Immune reconstitution inflammatory syndrome associated with pulmonary pathogens.

Immune reconstitution inflammatory syndrome (IRIS) is an exaggerated immune response to a variety of pathogens in response to antiretroviral therapy-mediated recovery of the immune system in HIV-infected patients. Although IRIS can occur in many organs, pulmonary IRIS, associated with opportunistic infections such as Mycobacterium tuberculosis and Pneumocystis jirovecii, is particularly associated with high morbidity and mortality. The pathology of IRIS is associated with a variety of innate and adaptive immune factors, including CD4+ T-cells, CD8+ T-cells, γδ T-cells, natural killer cells, macrophages, the complement system and surfactant proteins, Toll-like receptors and pro-inflammatory cytokines and chemokines. Although there are numerous reports about the immune factors involved in IRIS, the mechanisms involved in the development of pulmonary IRIS are poorly understood. Here, we propose that studies using gene-deficient murine and nonhuman primate models will help to identify the specific molecular targets associated with the development of IRIS. An improved understanding of the mechanisms involved in the pathology of pulmonary IRIS will help to identify potential biomarkers and therapeutic targets in this syndrome.

Neither classical nor alternative macrophage activation is required for Pneumocystis clearance during immune reconstitution inflammatory syndrome.

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4(+) T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2(-/-) mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2(-/-) mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR(-/-) nor RAG/IL-4Rα(-/-) mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR(-/-) mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα(-/-) mice. RAG/IFN-γR(-/-) mice had elevated numbers of lung CD4(+) T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8(+) T suppressor cells. Impaired lung CD8(+) T cell responses in RAG/IFN-γR(-/-) mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8(+) T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.

HIV-tuberculosis-associated immune reconstitution inflammatory syndrome is characterized by Toll-like receptor and inflammasome signalling.

Patients with HIV-associated tuberculosis (TB) initiating antiretroviral therapy (ART) may develop immune reconstitution inflammatory syndrome (TB-IRIS). No biomarkers for TB-IRIS have been identified and the underlying mechanisms are unclear. Here we perform transcriptomic profiling of the blood samples of patients with HIV-associated TB. We identify differentially abundant transcripts as early as week 0.5 post ART initiation that predict downstream activation of proinflammatory cytokines in patients who progress to TB-IRIS. At the characteristic time of TB-IRIS onset (week 2), the signature is characterized by over-representation of innate immune mediators including TLR signalling and TREM-1 activation of the inflammasome. In keeping with the transcriptional data, concentrations of plasma cytokines and caspase-1/5 are elevated in TB-IRIS. Inhibition of MyD88 adaptor and group 1 caspases reduces secretion of cytokines including IL-1 in TB-IRIS patients. These data provide insight on the pathogenesis of TB-IRIS and may assist the development of specific therapies.

Mycobacterial antigen driven activation of CD14++CD16- monocytes is a predictor of tuberculosis-associated immune reconstitution inflammatory syndrome.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14(++)CD16(-) monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment.

JCV GCN in a natalizumab-treated MS patient is associated with mutations of the VP1 capsid gene.

OBJECTIVE: To describe the clinical, neuroimaging, immunologic, and virologic characteristics of JC virus-associated granule cell neuronopathy (JCV GCN) in a natalizumab-treated patient with multiple sclerosis (MS) who developed immune reconstitution inflammatory syndrome (IRIS) after natalizumab withdrawal. METHODS: We obtained longitudinal clinical data as well as MRI and proton magnetic resonance spectroscopy from this patient with MS. We measured JCV-specific cellular immune response in his peripheral blood by intracellular cytokine staining and sequenced a fragment of JCV VP1 capsid gene detected in his CSF. We contrast our findings with the first recently reported case. RESULTS: This patient presented with worsening cerebellar symptoms and progressive cerebellar atrophy without new MS lesions on MRI after 63 months of natalizumab monotherapy. JCV DNA was detected in his CSF by PCR and harbored novel GCN-type mutations in the VP1 gene. He developed IRIS upon discontinuation of natalizumab and plasma exchange, which manifested itself by a worsening of clinical symptoms and contrast enhancement in the cerebellum on MRI. Treatment with corticosteroids resulted in resolution of IRIS, as demonstrated by proton magnetic resonance spectroscopy. The patient had a strong JCV-specific T-cell response in his peripheral blood and remains alive after 15 months from onset of symptoms, although with significant disability. He did not have MS relapse on glatiramer acetate. CONCLUSIONS: JCV GCN should be considered in patients on natalizumab presenting with progressive cerebellar symptoms and cerebellar atrophy, and is associated with mutations in the JCV VP1 gene. Natalizumab withdrawal may be complicated by JCV GCN IRIS, and require treatment with corticosteroids.

A non-synonymous polymorphism in IL-23R Gene (rs1884444) is associated with reduced risk to schistosomiasis-associated Immune Reconstitution Inflammatory Syndrome in a Kenyan population.

BACKGROUND: Human Immunodeficiency Virus (HIV) and Schistosomiasis co-infection is common among residents at the shores of Lake Victoria in Kenya. About 36% of this population initiating antiretroviral therapy (ART) experience Immune Reconstitution Inflammatory Syndrome (IRIS) that complicates recovery. Several IL-23R alleles have been associated with susceptibility to both autoimmune and inflammatory diseases through T-helper type 17 (TH17) cells. However, whether or not variants within the IL-23R increase susceptibility to IRIS in western Kenya is unknown. The objective of the current study was to determine the association between IL-23R gene polymorphisms, CD4+ cell counts and HIV RNA levels and IRIS in HIV and Schistosoma mansoni co-infected patients undergoing highly active anti-retroviral therapy (HAART). METHODS: A three-month case-control study was conducted on antiretroviral naïve schistosomiasis/HIV co-infected fishermen starting HAART in Uyoma Rarieda, Siaya County, Kenya. Seventy one patients were sampled at baseline and followed up for three months, to establish if they developed Schistosoma-related IRIS. In addition, the CD4+ cell counts and HIV RNA levels were determined in pre- and post-administration of HAART. Variations at five polymorphic sites of IL-23R (rs1884444, rs11465754, rs6682925, rs7530511 and rs7539625) based on >10% minor allele frequency in Yoruban reference population was determined using Allelic Discrimination Assay. The association between the five variants and susceptibility to IRIS was determined using logistic regression while controlling for potential confounders. In addition, the functional differences between the baseline CD4 + Cell counts and viral loads were determined using medians while across IL-23R genotypes were determined using Kruskal-Wallis tests. RESULTS: Overall, 26 (36.6%) patients developed schistosomiasis-associated IRIS at a median age of 35.5 years. Carriage of the TT genotype at the non-synonymous rs1884444 T > G relative to GG, was associated with a decreased risk of schistosomiasis-associated IRIS (OR, 0.25, 95% CI, 0.07-0.96, P = 0.043) while both baseline CD4+ cell counts and viral loads had no association with IRIS. CONCLUSION: These findings indicate that the non-synonymous variant rs1884444 T > G of IL-23R is associated with a decreased risk to schistosomiasis-associated IRIS. However, additional studies in a larger cohort and with an all inclusive polymorphic variants in the synonymous and non-synonymous regions need to be evaluated.

The role of monocytes in the development of Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome.

BACKGROUND: Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome (TB-IRIS) is a common complication of combined antiretroviral therapy (cART) in HIV-TB co-infected patients. However, the disease mechanism is poorly understood, prognosis of TB-IRIS is currently impossible, and diagnosis is highly challenging. We analyzed whether the gene expression of monocytes could be correlated with TB-IRIS pathogenesis and could be used to classify patients predisposed to TB-IRIS. METHODS: Monocyte gene expression was compared between patients who developed TB-IRIS and matched controls. We carried out whole-genome expression profiling using Affymetrix GeneChip(®) ST 1.1 arrays at two time-points: before cART initiation (baseline) and at week two post-cART initiation. For each time-point, we used different statistical approaches to identify molecular signatures which could be used as classifiers. We also functionally mapped the modulated cellular pathways using the software package Ingenuity Pathway Analysis. RESULTS: At baseline, before introduction of cART and before onset of symptoms, monocyte gene expression was already perturbed in patients who subsequently developed TB-IRIS, indicating a possible involvement of monocytes in TB-IRIS predisposition. The differences in monocyte gene expression in TB-IRIS patients became even more clear after two weeks of cART (when TB-IRIS commonly occurs), with more than 100 genes for which expression showed a fold change greater than 1.5. Both at baseline and at week two post-cART initiation, a classifier of 8 and 9 genes, respectively could be built, which allowed discrimination of TB-IRIS cases and controls. Pathway analyses revealed that the majority of the dysregulated genes in TB-IRIS - at the time of the IRIS episode, but also already at baseline - are associated with infection and inflammation. Relevant biological functions which were perturbed before/during TB-IRIS included "Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses" and "Complement System". CONCLUSION: Our results indicate an involvement of monocytes in predisposition to/development of TB-IRIS, and suggest a number of functional pathways which may play a role in TB-IRIS development. This comprehensive study of gene regulation in monocytes provides baseline data for further studies into biomarkers for prognosis and diagnosis of TB-IRIS.

The search for a genetic factor associating with immune restoration disease in HIV patients co-infected with Mycobacterium tuberculosis.

BACKGROUND: Up to 43% of HIV-infected patients co-infected with Mycobacterium tuberculosis experience exacerbations of tuberculosis (TB) after commencing antiretroviral therapy (ART). These are termed immune restoration disease (IRD). It is unclear why individual susceptibility varies. OBJECTIVE: We investigate if single nucleotide polymorphisms (SNP) in genes encoding cytokines, chemokines and their receptors associate with development of an IRD event in patients of two different ethnicities. METHODS: DNA samples were available from small well-characterised groups of HIV patients treated in Cambodia (TB-IRD, n=17; HIV(+)TB(+) controls, n=55) and India (TB-IRD, n=19; HIV(+)TB(+) controls, n= 43). HIV patients with a TB diagnosis but no evidence of IRD were included to control for susceptibility to TB per se. Sixteen SNP implicated in inflammation or mycobacterial disease were genotyped. RESULTS: Susceptibility to TB-IRD associated with carriage of TNFA-1031*T (rs1799964; P=0.05) and SLC11A1 D543N*G (rs17235409; P=0.04) in Cambodian patients and carriage of IL18-607*G (rs1946518; P=0.02) and VDR FokI (F/f)*T (rs10735810; P=0.05) in Indian patients. CONCLUSIONS: Associations between polymorphisms in immune-related genes and TB-IRD were found, but none were common across two ethnicities.