Immune mechanisms in fibrotic interstitial lung disease.

Fibrotic interstitial lung diseases (fILDs) have poor survival rates and lack effective therapies. Despite evidence for immune mechanisms in lung fibrosis, immunotherapies have been unsuccessful for major types of fILD. Here, we review immunological mechanisms in lung fibrosis that have the potential to impact clinical practice. We first examine innate immunity, which is broadly involved across fILD subtypes. We illustrate how innate immunity in fILD involves a complex interplay of multiple cell subpopulations and molecular pathways. We then review the growing evidence for adaptive immunity in lung fibrosis to provoke a re-examination of its role in clinical fILD. We close with future directions to address key knowledge gaps in fILD pathobiology: (1) longitudinal studies emphasizing early-stage clinical disease, (2) immune mechanisms of acute exacerbations, and (3) next-generation immunophenotyping integrating spatial, genetic, and single-cell approaches. Advances in these areas are essential for the future of precision medicine and immunotherapy in fILD.

Differential Responses to Targeting Matrix Metalloproteinase 9 in Idiopathic Pulmonary Fibrosis.

Rationale: Aberrant lung remodeling in idiopathic pulmonary fibrosis (IPF) is characterized by elevated MMP9 (matrix metalloproteinase 9) expression, but the precise role of this matrix metalloproteinase in this disease has yet to be fully elucidated.Objectives: To evaluate antifibrotic effects of MMP9 inhibition on IPF.Methods: Quantitative genomic, proteomic, and functional analyses both in vitro and in vivo were used to determine MMP9 expression in IPF cells and the effects of MMP9 inhibition on profibrotic mechanisms.Measurements and Main Results: In the present study, we demonstrate that MMP9 expression was increased in airway basal cell (ABC)-like cells from IPF lungs compared with ABC cells from normal lungs. The inhibition of MMP9 activity with an anti-MMP9 antibody, andecaliximab, blocked TGF-ß1 (transforming growth factor ß1)-induced Smad2 phosphorylation. However, in a subset of cells from patients with IPF, TGF-ß1 activation in their ABC-like cells was unaffected or enhanced by MMP9 blockade (i.e., nonresponders). Further analysis of nonresponder ABC-like cells treated with andecaliximab revealed an association with type 1 IFN expression, and the addition of IFNα to these cells modulated both MMP9 expression and TGF-ß1 activation. Finally, the inhibition of MMP9 ameliorated pulmonary fibrosis induced by responder lung cells but not a nonresponder in a humanized immunodeficient mouse model of IPF.Conclusions: Together, these data demonstrate that MMP9 regulates the activation of ABC-like cells in IPF and that targeting this MMP might be beneficial to a subset of patients with IPF who show sufficient expression of type 1 IFNs.

Modeling Idiopathic Pulmonary Fibrosis in Humanized Severe Combined Immunodeficient Mice.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease of unknown etiopathogenesis with limited therapeutic options. IPF is characterized by an abundance of fibroblasts and loss of epithelial progenitors, which cumulates in unrelenting fibrotic lung remodeling and loss of normal oxygenation. IPF has been challenging to model in rodents; nonetheless, mouse models of lung fibrosis provide clues as to the natural progression of lung injury and remodeling, but many have not been useful in predicting efficacy of therapeutics in clinical IPF. We provide a detailed methodologic description of various iterations of humanized mouse models, initiated by the i.v. injection of cells from IPF lung biopsy or explants specimens into severe combined immunodeficiency (SCID)/beige or nonobese diabetic SCID γ mice. Unlike cells from normal lung samples, IPF cells promote persistent, nonresolving lung remodeling in SCID mice. Finally, we provide examples and discuss potential advantages and pitfalls of human-specific targeting approaches in a humanized SCID model of pulmonary fibrosis.

Targeting of TAM Receptors Ameliorates Fibrotic Mechanisms in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling, which progressively abolishes lung function in an RTK (receptor tyrosine kinase)-dependent manner. Gas6 (growth arrest-specific 6) ligand, Tyro3 (TYRO3 protein tyrosine kinase 3), and Axl (anexelekto) RTK expression and activity are increased in IPF. OBJECTIVES: To determine if targeting these RTK pathways would inhibit fibroblast activation and the development of pulmonary fibrosis. METHODS: Quantitative genomic, proteomic, and functional analyses were used to determine Gas6/TAM (Tyro3, Axl, and Mertk [MER proto-oncogene, tyrosine kinase]) RTK expression and activation in tissues and fibroblasts from normal and IPF lungs. The profibrotic impact of these RTK pathways were also examined in bleomycin-induced pulmonary fibrosis and in SCID/Bg mice that developed pulmonary fibrosis after the intravenous administration of primary IPF fibroblasts. MEASUREMENTS AND MAIN RESULTS: Gas6, Axl, and Tyro3 were increased in both rapidly and slowly progressive IPF compared with normal lung samples and fibroblasts. Targeting these pathways with either specific antibodies directed at Gas6 or Axl, or with small-molecule TAM inhibitors indicated that the small molecule-mediated targeting approach was more efficacious in both in vitro and in vivo studies. Specifically, the TAM receptor inhibitor R428 (also known as BGB324) significantly inhibited the synthetic, migratory, and proliferative properties of IPF fibroblasts compared with the other Gas6/TAM receptor targeting agents. Finally, loss of Gas6 expression decreased lung fibrotic responses to bleomycin and treatment with R428 inhibited pulmonary fibrosis in humanized SCID/Bg mice. CONCLUSIONS: Gas6/TAM receptor activity contributes to the activation of pulmonary fibroblasts in IPF, suggesting that targeting this RTK pathway might be an effective antifibrotic strategy in this disease.

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis.

BACKGROUND: Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types. METHODS: Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441. RESULTS: DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells. CONCLUSIONS: Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Microbes Are Associated with Host Innate Immune Response in Idiopathic Pulmonary Fibrosis.

RATIONALE: Differences in the lung microbial community influence idiopathic pulmonary fibrosis (IPF) progression. Whether the lung microbiome influences IPF host defense remains unknown. OBJECTIVES: To explore the host immune response and microbial interaction in IPF as they relate to progression-free survival (PFS), fibroblast function, and leukocyte phenotypes. METHODS: Paired microarray gene expression data derived from peripheral blood mononuclear cells as well as 16S ribosomal RNA sequencing data from bronchoalveolar lavage obtained as part of the COMET-IPF (Correlating Outcomes with Biochemical Markers to Estimate Time-Progression in Idiopathic Pulmonary Fibrosis) study were used to conduct association pathway analyses. The responsiveness of paired lung fibroblasts to Toll-like receptor 9 (TLR9) stimulation by CpG-oligodeoxynucleotide (CpG-ODN) was integrated into microbiome-gene expression association analyses for a subset of individuals. The relationship between associated pathways and circulating leukocyte phenotypes was explored by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Down-regulation of immune response pathways, including nucleotide-binding oligomerization domain (NOD)-, Toll-, and RIG1-like receptor pathways, was associated with worse PFS. Ten of the 11 PFS-associated pathways correlated with microbial diversity and individual genus, with species accumulation curve richness as a hub. Higher species accumulation curve richness was significantly associated with inhibition of NODs and TLRs, whereas increased abundance of Streptococcus correlated with increased NOD-like receptor signaling. In a network analysis, expression of up-regulated signaling pathways was strongly associated with decreased abundance of operational taxonomic unit 1341 (OTU1341; Prevotella) among individuals with fibroblasts responsive to CpG-ODN stimulation. The expression of TLR signaling pathways was also linked to CpG-ODN responsive fibroblasts, OTU1341 (Prevotella), and Shannon index of microbial diversity in a network analysis. Lymphocytes expressing C-X-C chemokine receptor 3 CD8 significantly correlated with OTU1348 (Staphylococcus). CONCLUSIONS: These findings suggest that host-microbiome interactions influence PFS and fibroblast responsiveness.

Identification of promising plasma immune biomarkers to differentiate active pulmonary tuberculosis.

Although much research has been done related to biomarker discovery for tuberculosis infection, a set of biomarkers that can discriminate between active and latent TB diseases remains elusive. In the current study we correlate clinical aspects of TB disease with changes in the immune response as determined by biomarkers detected in plasma. Our study measured 18 molecules in human plasma in 17 patients with active disease (APTB), 14 individuals with latent tuberculosis infection (LTBI) and 16 uninfected controls (CTRL). We found that active tuberculosis patients have increased plasma levels of IL-6, IP-10, TNF-α, sCD163 and sCD14. Statistical analysis of these biomarkers indicated that simultaneous measurement of sCD14 and IL-6 was able to diagnose active tuberculosis infection with 83% accuracy. We also demonstrated that TNF-α and sCD163 were correlated with tuberculosis severity. We showed that the simultaneous detection of both plasma sCD14 and IL-6 is a promising diagnostic approach to identify APTB, and further, measurement of TNF-α and sCD163 can identify the most severe cases of tuberculosis.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Mycobacterium tuberculosis expressing phospholipase C subverts PGE2 synthesis and induces necrosis in alveolar macrophages.

BACKGROUND: Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. RESULTS: Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. CONCLUSIONS: Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.

Translational Studies Reveal the Divergent Effects of Simtuzumab Targeting LOXL2 in Idiopathic Pulmonary Fibrosis.

The composition of extracellular matrix (ECM) is altered during pathologic scarring in damaged organs including the lung. One major change in the ECM involves the cross-linking of collagen, which promotes fibroblast to myofibroblast differentiation. We examined the role of lysyl oxidase (LOX)-like 2 in lung progenitors and fibroblasts cultured from normal or IPF lung samples and in a humanized mouse model of IPF using a monoclonal antibody (Simtuzumab). Primary lung fibroblasts from normal donor lungs and IPF lung explants were examined for expression of LOXL2. Targeting LOXL2 with Simtuzumab on normal and IPF fibroblasts was examined both in vitro and in vivo for synthetic, functional, and profibrotic properties. LOXL2 was increased at transcript and protein level in IPF compared with normal lung samples. In a dose-dependent manner, Simtuzumab enhanced differentiation of fibroblasts into myofibroblasts. Inhibition of LOXL2 also enhanced fibroblast invasion and accelerated the outgrowth of fibroblasts from dissociated human lung cell preparations. Finally, preventative or delayed delivery of Simtuzumab enhanced lung fibrosis in a humanized mouse model of pulmonary fibrosis. Consistent with its failure in a Phase 2 clinical trial, Simtuzumab exhibited no therapeutic efficacy in translational in vitro and in vivo assays.

Central lung gene expression associates with myofibroblast features in idiopathic pulmonary fibrosis.

RATIONALE: Contribution of central lung tissues to pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unknown. OBJECTIVE: To ascertain the relationship between cell types of IPF-central and IPF-peripheral lung explants using RNA sequencing (RNA-seq) transcriptome. METHODS: Biopsies of paired IPF-central and IPF-peripheral along with non-IPF lungs were selected by reviewing H&E data. Criteria for differentially expressed genes (DEG) were set at false discovery rate <5% and fold change >2. Computational cell composition deconvolution was performed. Signature scores were computed for each cell type. FINDINGS: Comparison of central IPF versus non-IPF identified 1723 DEG (1522 upregulated and 201 downregulated). Sixty-two per cent (938/1522) of the mutually upregulated genes in central IPF genes were also upregulated in peripheral IPF versus non-IPF. Moreover, 85 IPF central-associated genes (CAG) were upregulated in central IPF versus both peripheral IPF and central non-IPF. IPF single-cell RNA-seq analysis revealed the highest CAG signature score in myofibroblasts and significantly correlated with a previously published activated fibroblasts signature (r=0.88, p=1.6×10-4). CAG signature scores were significantly higher in IPF than in non-IPF myofibroblasts (p=0.013). Network analysis of central-IPF genes identified a module significantly correlated with the deconvoluted proportion of myofibroblasts in central IPF and anti-correlated with inflammation foci trait in peripheral IPF. The module genes were over-represented in idiopathic pulmonary fibrosis signalling pathways. INTERPRETATION: Gene expression in central IPF lung regions demonstrates active myofibroblast features that contributes to disease progression. Further elucidation of pathological transcriptomic state of cells in the central regions of the IPF lung that are relatively spared from morphological rearrangements may provide insights into molecular changes in the IPF progression.

Antibody-mediated depletion of CCR10+EphA3+ cells ameliorates fibrosis in IPF.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.

Characterization of CD28null T cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease, with unknown etiopathogenesis and suboptimal therapeutic options. Previous reports have shown that increased T-cell numbers and CD28null phenotype is predictive of prognosis in IPF, suggesting that these cells might have a role in this disease. Flow cytometric analysis of explanted lung cellular suspensions showed a significant increase in CD8+ CD28null T cells in IPF relative to normal lung explants. Transcriptomic analysis of CD3+ T cells isolated from IPF lung explants revealed a loss of CD28-transcript expression and elevation of pro-inflammatory cytokine expression in IPF relative to normal T cells. IPF lung explant-derived T cells (enriched with CD28null T cells), but not normal donor lung CD28+ T cells induced dexamethasone-resistant lung remodeling in humanized NSG mice. Finally, CD28null T cells expressed similar CTLA4 and significantly higher levels of PD-1 proteins relative to CD28+ T cells and blockade of either proteins in humanized NSG mice, using anti-CTLA4, or anti-PD1, mAb treatment-accelerated lung fibrosis. Together, these results demonstrate that IPF CD28null T cells may promote lung fibrosis but the immune checkpoint proteins, CTLA-4 and PD-1, appears to limit this effect.

Targeting MAP3K19 prevents human lung myofibroblast activation both in vitro and in a humanized SCID model of idiopathic pulmonary fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-ß pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

CCR10+ epithelial cells from idiopathic pulmonary fibrosis lungs drive remodeling.

Idiopathic pulmonary fibrosis (IPF) is a devastating fibrotic lung disease of unknown etiology and limited therapeutic options. In this report, we characterize what we believe is a novel CCR10+ epithelial cell population in IPF lungs. There was a significant increase in the percentage of CCR10+ epithelial cells in IPF relative to normal lung explants and their numbers significantly correlated to lung remodeling in humanized NSG mice. Cultured CCR10-enriched IPF epithelial cells promoted IPF lung fibroblast invasion and collagen 1 secretion. Single-cell RNA sequencing analysis showed distinct CCR10+ epithelial cell populations enriched for inflammatory and profibrotic transcripts. Consistently, cultured IPF but not normal epithelial cells induced lung remodeling in humanized NSG mice, where the number of CCR10+ IPF, but not normal, epithelial cells correlated with hydroxyproline concentration in the remodeled NSG lungs. A subset of IPF CCR10hi epithelial cells coexpress EphA3 and ephrin A signaling induces the expression of CCR10 by these cells. Finally, EphA3+CCR10hi epithelial cells induce more consistent lung remodeling in NSG mice relative to EphA3-CCR10lo epithelial cells. Our results suggest that targeting epithelial cells, highly expressing CCR10, may be beneficial in IPF.

CD18 Regulates Monocyte Hematopoiesis and Promotes Resistance to Experimental Schistosomiasis.

Infection with Schistosoma mansoni causes a chronic parasitic disease that progress to severe liver and gastrointestinal damage, and eventually death. During its development into mammalian hosts, immature schistosomula transit through the lung vasculature before they reach the liver to mature into adult worms. A low grade inflammatory reaction is induced during this process. However, molecules that are required for efficient leukocyte accumulation in the lungs of S. mansoni-infected subjects are unknown. In addition, specific leukocyte subsets that mediate pulmonary response during S. mansoni migration through the lung remain to be elucidated. ß2 integrins are fundamental regulators of leukocyte trans-endothelial migration and function. Therefore, we investigated their role during experimental schistosomiasis. Mice that express low levels of CD18 (the common ß2 integrin subunit) and wild type C57BL/6 mice were subcutaneously infected with S. mansoni cercariae. Cellular profiles of lungs and livers were evaluated in different time points after infection by flow cytometry. Low levels of CD18 affected the accumulation of patrolling Ly6Clow, intermediate Ly6Cinter monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells in the lungs 7 days after infection. This correlated with increased TNF-α levels. Strikingly, low CD18 expression resulted in monocytopenia both in the peripheral blood and bone marrow during acute infection. After 48 days, S. mansoni worm burdens were higher in the hepatic portal system of CD18low mice, which also displayed reduced hepatic accumulation of patrolling Ly6Clow and intermediate Ly6Cinter, but not inflammatory Ly6Chigh monocytes. Higher parasite burden resulted in increased granulomatous lesions in the liver, increased egg deposition and enhanced mortality. Overall, our data point for a fundamental role of CD18 for monocyte hematopoiesis during infection, which promotes an efficient host response against experimental schistosomiasis.

Epigenetic alterations are associated with monocyte immune dysfunctions in HIV-1 infection.

Monocytes are key cells in the immune dysregulation observed during human immunodeficiency virus (HIV) infection. The events that take place specifically in monocytes may contribute to the systemic immune dysfunction characterized by excessive immune activation in infected individuals, which directly correlates with pathogenesis and progression of the disease. Here, we investigated the immune dysfunction in monocytes from untreated and treated HIV + patients and associated these findings with epigenetic changes. Monocytes from HIV patients showed dysfunctional ability of phagocytosis and killing, and exhibited dysregulated cytokines and reactive oxygen species production after M. tuberculosis challenge in vitro. In addition, we showed that the expression of enzymes responsible for epigenetic changes was altered during HIV infection and was more prominent in patients that had high levels of soluble CD163 (sCD163), a newly identified plasmatic HIV progression biomarker. Among the enzymes, histone acetyltransferase 1 (HAT1) was the best epigenetic biomarker correlated with HIV - sCD163 high patients. In conclusion, we confirmed that HIV impairs effector functions of monocytes and these alterations are associated with epigenetic changes that once identified could be used as targets in therapies aiming the reduction of the systemic activation state found in HIV patients.

Classical and alternative macrophages have impaired function during acute and chronic HIV-1 infection.

OBJECTIVES: Three decades after HIV recognition and its association with AIDS development, many advances have emerged - especially related to prevention and treatment. Undoubtedly, the development of Highly Active Antiretroviral Therapy (HAART) dramatically changed the future of the syndrome that we know today. In the present study, we evaluate the impact of Highly Active Antiretroviral Therapy on macrophage function and its relevance to HIV pathogenesis. METHODS: PBMCs were isolated from blood samples and monocytes (CD14+ cells) were purified. Monocyte-Derived Macrophages (MDMs) were activated on classical (MGM-CSF+IFN-γ) or alternative (MIL-4+IL13) patterns using human recombinant cytokines for six days. After this period, Monocyte-Derived Macrophages were stimulated with TLR2/Dectin-1 or TLR4 agonists and we evaluated the influence of HIV-1 infection and Highly Active Antiretroviral Therapy on the release of cytokines/chemokines by macrophages. RESULTS: The data were obtained using Monocyte-Derived Macrophages derived from HIV naïve or from patients on regular Highly Active Antiretroviral Therapy. Classically Monocyte-Derived Macrophages obtained from HIV-1 infected patients on Highly Active Antiretroviral Therapy released higher levels of IL-6 and IL-12 even without PAMPs stimuli when compared to control group. On the other hand, alternative Monocyte-Derived Macrophages derived from HIV-1 infected patients on Highly Active Antiretroviral Therapy released lower levels of IL-6, IL-10, TNF-α, IP-10 and RANTES after LPS stimuli when compared to control group. Furthermore, healthy individuals have a complex network of cytokines/chemokines released by Monocyte-Derived Macrophages after PAMP stimuli, which was deeply affected in MDMs obtained from naïve HIV-1 infected patients and only partially restored in MDMs derived from HIV-1 infected patients even on regular Highly Active Antiretroviral Therapy. CONCLUSION: Our therapy protocols were not effective in restoring the functional alterations induced by HIV, especially those found on macrophages. These findings indicate that we still need to develop new approaches and improve the current therapy protocols, focusing on the reestablishment of cellular functions and prevention/treatment of opportunistic infections.