Araceli Valverde

Dendritic cells (DCs) are central orchestrators of adaptive immunity, integrating innate immune sensing with antigen‐specific T‐cell activation. While long noncoding RNAs (lncRNAs) have emerged as critical regulators of immune cell fate and function, the lncRNA landscape governing human DC differentiation and effector activity remains incompletely defined. Here, we generated monocyte‐derived dendritic cells (moDCs) from primary human peripheral blood monocytes and performed transcriptome‐wide RNA sequencing to systematically identify previously unannotated lncRNAs associated with DC differentiation. We uncovered an extensive repertoire of novel lncRNA transcripts dynamically regulated during DC development. Among these, MSTRG.479558.1 and MSTRG.582898.5 were prioritized based on robust induction during differentiation, responsiveness to inflammatory stimulation (LPS), and elevated expression in periodontitis. Functional interrogation revealed a selective and non‐redundant role for MSTRG.479558.1 in DC biology. Knockdown of the lncRNA MSTRG.479558.1 but not MSTRG.582898.5 significantly reduced the expression of dendritic cell markers CD1a, CD209, and CDw93, indicating impaired dendritic cell differentiation. Functionally, MSTRG.479558.1 silencing profoundly impaired phagocytic capacity and antigen processing, indicating disruption at the earliest stages of antigen handling. Moreover, loss of MSTRG.479558.1 compromised antigen presentation and significantly attenuated DC‐mediated CD4 ⁺ T‐cell activation, and Th1 related cytokines IFNγ and TNF‐α, suggesting a functional link between this lncRNA and adaptive immune priming. Mechanistically, targeted transcriptional profiling demonstrated coordinated dysregulation of genes implicated in antigen processing and presentation pathways following MSTRG.479558.1 depletion, including components of proteasomal processing, chaperone‐mediated folding, and MHC‐associated peptide loading machinery. These findings support a model in which MSTRG.479558.1 acts as an upstream regulator of a transcriptional network that governs antigen handling and presentation. Collectively, this study defines a previously uncharacterized lncRNA program operative during human DC differentiation and identifies MSTRG.479558.1 as a critical regulator of DC functional competency. By coupling antigen acquisition to T‐cell activation, this lncRNA represents a novel layer of immune regulation with potential implications for inflammatory disease pathogenesis and therapeutic targeting.

Bone remodeling relies on the coordinated activity of osteoblasts (OBs) and osteoclasts (OCs). Disruptions in OB-OC balance can lead to diseases such as periodontitis, a chronic microbial-induced inflammatory disease. To investigate how inflammation affects OB-OC interactions, we standardized an in vitro 2D indirect co-culture system using primary human OB and OC precursors from peripheral blood mononuclear cells in a transwell setup, which allows paracrine signaling and separate analysis of each cell type. When exposed to bacterial lipopolysaccharides (Aa LPS and E. coli LPS) and proinflammatory cytokines (IL-6 and TNF-α), we observed that inflammatory stimuli significantly increased OC differentiation, particularly TNF-α, while E. coli LPS specifically suppressed OB activity as observed by the expression of key markers and cellular staining. These results demonstrate that microbial and host-derived inflammatory factors can differentially modulate bone cell behavior. This approach offers a physiologically relevant and ethically advantageous alternative to animal models to screen dual-targeted bone therapies to restore perturbed metabolism.

Aims: Elevated levels of Herpes Simplex Virus 1 (HSV‐1) have been reported in periodontitis, however, the tropism and relationship with periodontal inflammation are poorly characterized. This study investigated how inflammation affects viral tropism toward human periodontal ligament stem cells (hPDLSCs). Methods: HSV‐1 gB and gD transcripts in healthy and diseased human gingiva were measured by RT‐qPCR and confirmed in HSV‐1‐infected murine gingiva. HSV‐1 infection in hPDLSCs was analyzed by imaging and flow cytometry. hPDLSCs were individually treated with IL‐6, TNF‐α, GMCSF, IL‐10, or PgLPS, and HSV‐1 replication was assessed by infecting with the 17 GFP strain. Lineage markers in virally infected hPDLSCs during osteogenic differentiation were measured by RT‐qPCR and immunofluorescence in vitro and validated in vivo. Mice subjected to ligature‐induced periodontitis (LIP) and infected with HSV‐1 were examined for gingival histology, inflammatory cytokines, and alveolar bone loss. Results: Inflamed human gingiva showed higher expression of viral transcripts compared to healthy controls. In mouse oral HSV‐1 infection, gB and gD expression increased over time, with higher levels in mice with ligature‐induced periodontitis. Virus infected hPDLSCs challenged with inflammatory mediators or PgLPS showed higher GFP, while IL‐10 treatment attenuated GFP levels. Importantly, HSV‐1 17 GFP infection affected osteoblast lineage commitment by promoting the expression of key transcription factors in vitro and in vivo. Compared to the LIP alone group, higher levels of inflammatory markers and bone loss were evident in HSV‐1 infected with LIP. Conclusion: hPDLSCs are trophic to HSV‐1 in vitro and in vivo, with periodontal inflammation playing a significant role in viral tropism.

Aim: Polarization of macrophages (Mφ) is a well‐controlled axis with considerable consequences in both the pro‐inflammatory and resolution phases of inflammation. We aimed to determine if periodontal therapy may instigate M1 to M2 Mφ polarization favoring resolution of inflammation within periodontal tissues.Methods: Gingival biopsies were excised from subjects diagnosed with Stage III, Grade B periodontitis before and 4–6 weeks after nonsurgical periodontal therapy. Total RNA was isolated and pro‐ and anti‐inflammatory markers associated with Mφ polarization assessed by RT‐qPCR. Mice were subject to ligature‐induced periodontitis and gingival tissues collected after 8 days in‐situ or 10 days after ligature removal and M1 and M2 Mφ markers examined by RT‐qPCR and flow cytometry.Results: In human samples, improvement in clinical parameters posttherapy correlates with reduced bacterial burden, downregulation in M1 (TNF‐α, STAT1, CXCL10, and miR‐155), and elevated levels of M2 (STAT6, TGM2, CCL22, and IL‐10) Mφ markers. In a murine model of resolution of LIP, we observed reduced levels of M1 Mφ markers cox2, iNOS2, F4/80+CD80+, and F4/80+CD86+ and elevated levels of M2‐like Mφ markers tgm2, arg1, F4/80+CD206+ and F4/80+CD163+ corroborated human findings.Conclusion: Resolution of periodontal inflammation is associated with M1 to M2 Mφ polarization after nonsurgical periodontal therapy. Assessment of Mφ markers can provide relevant clinical information on the successful response of periodontal therapy and may be used to target nonresponders.

Periodontitis is a multifactorial immune‐mediated disease exacerbated by dysregulated alveolar bone homeostasis. Timely intervention is crucial for disease management to prevent tooth loss. To successfully manage periodontitis, it is imperative to understand the cellular and molecular mechanisms involved in its pathogenesis to develop novel treatment modalities. Non‐surgical periodontal therapy (NSPT) such as subgingival instrumentation/debridement has been the underlying treatment strategy over the past decades. However, new NSPT approaches that target key signaling pathways regulating alveolar bone homeostasis have shown positive clinical outcomes. This narrative review aims to discuss endogenous bone homeostasis mechanisms impaired in periodontitis and highlight the clinical outcomes of preventive periodontal therapy to avoid invasive periodontal therapies. Although the anti‐resorptive therapeutic adjuncts have demonstrated beneficial outcomes, adverse events have been reported. Diverse immunomodulatory therapies targeting the osteoblast/osteoclast (OB/OC) axis have shown promising outcomes in vivo. Future controlled randomized clinical trials (RCT) would help clinicians and patients in the selection of novel preventing therapies targeting key molecules to effectively treat or prevent periodontitis.

Introduction Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated. Objective To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses. Methods Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation, polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs, M1/M2 marker expression. Results Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarization in vivo. Conclusion Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.

Introduction: Macrophages (Mφ) can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, and tissue regeneration. Noncoding RNAs play critical roles in numerous biological pathways; however, their functional interaction in the regulation of Mφ polarization and immune responses remain unclear. Objectives: To examine relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. Methods: Expression of MALAT1 and miR-30b was examined in differentiating M1/M2 Mφ, human and murine inflamed gingival biopsies by RT-qPCR. MALAT1 and miR-30b direct interaction was examined by dual luciferase assays. Impact of MALAT1 knockdown and miR-30b overexpression was examined on macrophage polarization markers, bacterial phagocytosis, antigen uptake/processing and cytokine profiles. Results: MALAT1 expression displays a time-dependent induction during Mφ differentiation and, upon challenge with TLR4 agonist ( E. coli LPS). Knockdown of MALAT1 enhanced the expression of M2Mφ markers without affecting the M1Mφ markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 polarization. MALAT1 knockdown Mφ exhibit reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2Mφ differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, and downregulation of miR-30b that correlates with higher M1Mφ markers expression in gingival tissues suggesting a pro-inflammatory function of MALAT1. Conclusion: MALAT1/miR-30b antagonistic interaction shapes Mφ polarization in vitro and in inflamed gingival biopsies.

Long noncoding RNA (lncRNA) are a class of endogenous, non-protein coding RNAs that are increasingly being associated with various cellular functions and diseases. Yet, despite their ubiquity and abundance, only a minute fraction of these molecules has an assigned function. LncRNAs show tissue-, cell-, and developmental stage-specific expression, and are differentially expressed under physiological or pathological conditions. The role of lncRNAs in the lineage commitment of immune cells and shaping immune responses is becoming evident. Myeloid cells and lymphoid cells are two major classes of immune systems that work in concert to initiate and amplify innate and adaptive immunity in vertebrates. In this review, we provide mechanistic roles of lncRNA through which these noncoding RNAs can directly participate in the differentiation, polarization, and activation of myeloid (monocyte, macrophage, and dendritic cells) and lymphoid cells (T cells, B cells, and NK cells). While our knowledge on the role of lncRNA in immune cell differentiation and function has improved in the past decade, further studies are required to unravel the biological role of lncRNAs and identify novel mechanisms of lncRNA functions in immune cells. Harnessing the regulatory potential of lncRNAs can provide novel diagnostic and therapeutic targets in treating immune cell related diseases.