PRMT5 Promotes T follicular helper Cell Differentiation and Germinal Center Responses during Influenza Virus Infection.

Protein arginine methyltransferases (PRMTs) modify diverse protein targets and regulate numerous cellular processes; yet, their contributions to individual effector T cell responses during infections are incompletely understood. In this study, we identify PRMT5 as a critical regulator of CD4+ T follicular helper cell (Tfh) responses during influenza virus infection in mice. Conditional PRMT5 deletion in murine T cells results in an almost complete ablation of both Tfh and T follicular regulatory populations and, consequently, reduced B cell activation and influenza-specific Ab production. Supporting a potential mechanism, we observe elevated surface expression of IL-2Rα on non-T regulatory effector PRMT5-deficient T cells. Notably, IL-2 signaling is known to negatively impact Tfh differentiation. Collectively, our findings identify PRMT5 as a prominent regulator of Tfh programming, with potential causal links to IL-2 signaling.

Sterols in asthma.

While sterols regulate immune processes key to the pathogenesis of asthma, inhibition of sterols with statin drugs has shown conflicting results in human asthma. Here, a novel understanding of the impact of sterols on type 17 immune responses and asthma lead us to hypothesize that sterols and statins may be relevant to severe asthma endotypes with neutrophil infiltration.

The Minority Scientists' Experience: Challenging and Overcoming Barriers to Enhancing Diversity and Career Advancement.

Minority groups face barriers in accessing quality health care, professional advancement, and representation in immunology research efforts as a result of institutional racism that if unaddressed can perpetuate a lack of diversity. In 2021, the AAI Minority Affairs Committee convened a cross section of academic and industry scientists from underrepresented groups at various stages of their professions to discuss how best to address the toll racism takes on study design and scientific careers. Panelists drew directly from their own experiences as scientists to share perspectives and strategies for countering a lack of representation in clinical research, responding to microaggressions, navigating academic advancement, and providing effective mentorship. The session reinforced the need for minority scientists to take an active role in advocating for diversity, engaging mentors, and taking responsibility to face rather than avoid institutional obstacles. Overall, increased dialogue and institutional awareness of the experience of scientists from underrepresented groups in research remain the best tools to ensure a health equity mindset and advancement of their careers.

PRMT5 in T Cells Drives Th17 Responses, Mixed Granulocytic Inflammation, and Severe Allergic Airway Inflammation.

Severe asthma is characterized by steroid insensitivity and poor symptom control and is responsible for most asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding of mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in human asthmatic lungs. In this study, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling, and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, retinoic acid-related orphan receptor γt (RORγt), and Th17 responses, with PRMT5-dependent increases in RORγt's agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.

Sterols and immune mechanisms in asthma.

The field of sterol and oxysterol biology in lung disease has recently gained attention, revealing a unique need for sterol uptake and metabolism in the lung. The presence of cholesterol transport, biosynthesis, and sterol/oxysterol-mediated signaling in immune cells suggests a role in immune regulation. In support of this idea, statin drugs that inhibit the cholesterol biosynthesis rate-limiting step enzyme, hydroxymethyl glutaryl coenzyme A reductase, show immunomodulatory activity in several models of inflammation. Studies in human asthma reveal contradicting results, whereas promising retrospective studies suggest benefits of statins in severe asthma. Here, we provide a timely review by discussing the role of sterols in immune responses in asthma, analytical tools to evaluate the role of sterols in disease, and potential mechanistic pathways and targets relevant to asthma. Our review reveals the importance of sterols in immune processes and highlights the need for further research to solve critical gaps in the field.

Protein Arginine Methyltransferase 5 in T Lymphocyte Biology.

Protein arginine methyltransferase 5 (PRMT5) is the major methyltransferase (MT) catalyzing symmetric dimethylation (SDM). PRMT5 regulates developmental, homeostatic and disease processes in vertebrates and invertebrates, and a carcinogenic role has been observed in mammals. Recently, tools generated for PRMT5 loss of function have allowed researchers to demonstrate essential roles for PRMT5 in mouse and human lymphocyte biology. PRMT5 modulates CD4+ and CD8+ T cell development in the thymus, peripheral homeostasis, and differentiation into CD4+ helper T lymphocyte (Th)17 cell phenotypes. Here, we provide a timely review of the milestones leading to our current understanding of PRMT5 in T cell biology, discuss current tools to modify PRMT5 expression/activity, and highlight mechanistic pathways.

CD8+ T cells contribute to diet-induced memory deficits in aged male rats.

We have previously shown that short-term (3-day) high fat diet (HFD) consumption induces a neuroinflammatory response and subsequent impairment of long-term memory in aged, but not young adult, male rats. However, the immune cell phenotypes driving this proinflammatory response are not well understood. Previously, we showed that microglia isolated from young and aged rats fed a HFD express similar levels of priming and proinflammatory transcripts, suggesting that additional factors may drive the exaggerated neuroinflammatory response selectively observed in aged HFD-fed rats. It is established that T cells infiltrate both the young and especially the aged central nervous system (CNS) and contribute to immune surveillance of the parenchyma. Thus, we investigated the modulating role of short-term HFD on T cell presence in the CNS in aged rats using bulk RNA sequencing and flow cytometry. RNA sequencing results indicate that aging and HFD altered the expression of genes and signaling pathways associated with T cell signaling, immune cell trafficking, and neuroinflammation. Moreover, flow cytometry data showed that aging alone increased CD4+ and CD8+ T cell presence in the brain and that CD8+, but not CD4+, T cells were further increased in aged rats fed a HFD. Based on these data, we selectively depleted circulating CD8+ T cells via an intravenous injection of an anti-CD8 antibody in aged rats prior to 3 days of HFD to infer the functional role these cells may be playing in long-term memory and neuroinflammation. Results indicate that peripheral depletion of CD8+ T cells lowered hippocampal cytokine levels and prevented the HFD-induced i) increase in brain CD8+ T cells, ii) memory impairment, and iii) alterations in pre- and post-synaptic structures in the hippocampus and amygdala. Together, these data indicate a substantial role for CD8+ T cells in mediating diet-induced memory impairments in aged male rats.

Corticosteroid insensitivity persists in the absence of STAT1 signaling in severe allergic airway inflammation.

Corticosteroid insensitivity in asthma limits the ability to effectively manage severe asthma, which is characterized by persistent airway inflammation, airway hyperresponsiveness (AHR), and airflow obstruction despite corticosteroid treatment. Recent reports indicate that corticosteroid insensitivity is associated with increased interferon-γ (IFN-γ) levels and T-helper (Th) 1 lymphocyte infiltration in severe asthma. Signal transducer and activator of transcription 1 (STAT1) activation by IFN-γ is a key signaling pathway in Th1 inflammation; however, its role in the context of severe allergic airway inflammation and corticosteroid sensitivity remains unclear. In this study, we challenged wild-type (WT) and Stat1-/- mice with mixed allergens (MA) augmented with c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate], an inducer of Th1 cell infiltration with increased eosinophils, neutrophils, Th1, Th2, and Th17 cells. Compared with WT mice, Stat1-/- had reduced neutrophils, Th1, and Th17 cell infiltration. To evaluate corticosteroid sensitivity, mice were treated with either vehicle, 1 or 3 mg/kg fluticasone propionate (FP). Corticosteroids significantly reduced eosinophil infiltration and cytokine levels in both c-di-GMP + MA-challenged WT and Stat1-/- mice. However, histological and functional analyses show that corticosteroids did not reduce airway inflammation, epithelial mucous cell abundance, airway smooth muscle mass, and AHR in c-di-GMP + MA-challenged WT or Stat1-/- mice. Collectively, our data suggest that increased Th1 inflammation is associated with a decrease in corticosteroid sensitivity. However, increased airway pathology and AHR persist in the absence of STAT1 indicate corticosteroid insensitivity in structural airway cells is a STAT1 independent process.

Dexamethasone and Fumaric Acid Ester Conjugate Synergistically Inhibits Inflammation and NF-κB in Macrophages.

Macrophage-mediated inflammation drives autoimmune and chronic inflammatory diseases. Treatment with anti-inflammatory agents can be an effective strategy to reduce this inflammation; however, high concentrations of these agents can have immune-dampening and other serious side effects. Synergistic combination of anti-inflammatory agents can mitigate dosing by requiring less drug. Multiple anti-inflammatory agents were evaluated in combination for synergistic inhibition of macrophage inflammation. The most potent synergy was observed between dexamethasone (DXM) and fumaric acid esters (e.g., monomethyl fumarate (MMF)). Furthermore, this combination was found to synergistically inhibit inflammatory nuclear factor κB (NF-κB) transcription factor activity. The optimal ratio for synergy was determined to be 1:1, and DXM and MMF were conjugated by esterification at this molar ratio. The DXM-MMF conjugate displayed improved inhibition of inflammation over the unconjugated combination in both murine and human macrophages. In the treatment of human donor monocyte-derived macrophages, the combination of DXM and MMF significantly inhibited inflammatory gene expression downstream of NF-κB and overall performed better than either agent alone. Further, the DXM-MMF conjugate significantly inhibited expression of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome-associated genes. The potent anti-inflammatory activity of the DXM-MMF conjugate in human macrophages indicates that it may have benefits in the treatment of autoimmune and inflammatory diseases.

MicroRNA 155 Contributes to Host Immunity against Leishmania donovani but Is Not Essential for Resolution of Infection.

CD4+ T helper 1 (Th1) cells producing interferon gamma (IFN-γ) are critical for the resolution of visceral leishmaniasis (VL). MicroRNA 155 (miR155) promotes CD4+ Th1 responses and IFN-γ production by targeting suppressor of cytokine signaling-1 (SOCS1) and Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1) and therefore could play a role in the resolution of VL. To determine the role of miR155 in VL, we monitored the course of Leishmania donovani infection in miR155 knockout (miR155KO) and wild-type (WT) C57BL/6 mice. miR155KO mice displayed significantly higher liver and spleen parasite loads than WT controls and showed impaired hepatic granuloma formation. However, parasite growth eventually declined in miR155KO mice, suggesting the induction of a compensatory miR155-independent antileishmanial pathway. Leishmania antigen-stimulated splenocytes from miR155KO mice produced significantly lower levels of Th1-associated IFN-γ than controls. Interestingly, at later time points, levels of Th2-associated interleukin-4 (IL-4) and IL-10 were also lower in miR155KO splenocyte supernatants than in WT mice. On the other hand, miR155KO mice displayed significantly higher levels of IFN-γ, iNOS, and TNF-α gene transcripts in their livers than WT mice, indicating that distinct organ-specific antiparasitic mechanisms were involved in control of L. donovani infection in miR155KO mice. Throughout the course of infection, organs of miR155KO mice showed significantly more PDL1-expressing Ly6Chi inflammatory monocytes than WT mice. Conversely, blockade of Ly6Chi inflammatory monocyte recruitment in miR155KO mice significantly reduced parasitic loads, indicating that these cells contributed to disease susceptibility. In conclusion, we found that miR155 contributes to the control of L. donovani but is not essential for infection resolution.

PRMT5-Selective Inhibitors Suppress Inflammatory T Cell Responses and Experimental Autoimmune Encephalomyelitis.

In the autoimmune disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), expansion of pathogenic, myelin-specific Th1 cell populations drives active disease; selectively targeting this process may be the basis for a new therapeutic approach. Previous studies have hinted at a role for protein arginine methylation in immune responses, including T cell-mediated autoimmunity and EAE. However, a conclusive role for the protein arginine methyltransferase (PRMT) enzymes that catalyze these reactions has been lacking. PRMT5 is the main PRMT responsible for symmetric dimethylation of arginine residues of histones and other proteins. PRMT5 drives embryonic development and cancer, but its role in T cells, if any, has not been investigated. In this article, we show that PRMT5 is an important modulator of CD4+ T cell expansion. PRMT5 was transiently upregulated during maximal proliferation of mouse and human memory Th cells. PRMT5 expression was regulated upstream by the NF-κB pathway, and it promoted IL-2 production and proliferation. Blocking PRMT5 with novel, highly selective small molecule PRMT5 inhibitors severely blunted memory Th expansion, with preferential suppression of Th1 cells over Th2 cells. In vivo, PRMT5 blockade efficiently suppressed recall T cell responses and reduced inflammation in delayed-type hypersensitivity and clinical disease in EAE mouse models. These data implicate PRMT5 in the regulation of adaptive memory Th cell responses and suggest that PRMT5 inhibitors may be a novel therapeutic approach for T cell-mediated inflammatory disease.

miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair.

UNLABELLED: Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers including inflammation. microRNA (miR)-155-5p is a small, noncoding RNA that negatively regulates mRNA translation. In macrophages, miR-155-5p is induced by inflammatory stimuli and elicits a response that could be toxic after SCI. miR-155 may also independently alter expression of genes that regulate axon growth in neurons. Here, we hypothesized that miR-155 deletion would simultaneously improve axon growth and reduce neuroinflammation after SCI by acting on both neurons and macrophages. New data show that miR-155 deletion attenuates inflammatory signaling in macrophages, reduces macrophage-mediated neuron toxicity, and increases macrophage-elicited axon growth by ∼40% relative to control conditions. In addition, miR-155 deletion increases spontaneous axon growth from neurons; adult miR-155 KO dorsal root ganglion (DRG) neurons extend 44% longer neurites than WT neurons. In vivo, miR-155 deletion augments conditioning lesion-induced intraneuronal expression of SPRR1A, a regeneration-associated gene; ∼50% more injured KO DRG neurons expressed SPRR1A versus WT neurons. After dorsal column SCI, miR-155 KO mouse spinal cord has reduced neuroinflammation and increased peripheral conditioning-lesion-enhanced axon regeneration beyond the epicenter. Finally, in a model of spinal contusion injury, miR-155 deletion improves locomotor function at postinjury times corresponding with the arrival and maximal appearance of activated intraspinal macrophages. In miR-155 KO mice, improved locomotor function is associated with smaller contusion lesions and decreased accumulation of inflammatory macrophages. Collectively, these data indicate that miR-155 is a novel therapeutic target capable of simultaneously overcoming neuron-intrinsic and neuron-extrinsic barriers to repair after SCI. SIGNIFICANCE STATEMENT: Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers, including inflammation. Here, new data show that deleting microRNA-155 (miR-155) affects both mechanisms and improves repair and functional recovery after SCI. Macrophages lacking miR-155 have altered inflammatory capacity, which enhances neuron survival and axon growth of cocultured neurons. In addition, independent of macrophages, adult miR-155 KO neurons show enhanced spontaneous axon growth. Using either spinal cord dorsal column crush or contusion injury models, miR-155 deletion improves indices of repair and recovery. Therefore, miR-155 has a dual role in regulating spinal cord repair and may be a novel therapeutic target for SCI and other CNS pathologies.

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

MicroRNAs targeting TGFß signalling underlie the regulatory T cell defect in multiple sclerosis.

Transforming growth factor beta (TGFß) signalling is critical for regulatory T cell development and function, and regulatory T cell dysregulation is a common observation in autoimmune diseases, including multiple sclerosis. In a comprehensive miRNA profiling study of patients with multiple sclerosis naïve CD4 T cells, 19 differentially expressed miRNAs predicted to target the TGFß signalling pathway were identified, leading to the hypothesis that miRNAs may be responsible for the regulatory T cell defect observed in patients with multiple sclerosis. Patients with multiple sclerosis had reduced levels of TGFß signalling components in their naïve CD4 T cells. The differentially expressed miRNAs negatively regulated the TGFß pathway, resulting in a reduced capacity of naïve CD4 T cells to differentiate into regulatory T cells. Interestingly, the limited number of regulatory T cells, that did develop when these TGFß-targeting miRNAs were overexpressed, were capable of suppressing effector T cells. As it has previously been demonstrated that compromising TGFß signalling results in a reduced regulatory T cell repertoire insufficient to control autoimmunity, and patients with multiple sclerosis have a reduced regulatory T cell repertoire, these data indicate that the elevated expression of multiple TGFß-targeting miRNAs in naïve CD4 T cells of patients with multiple sclerosis impairs TGFß signalling, and dampens regulatory T cell development, thereby enhancing susceptibility to developing multiple sclerosis.

Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells.

BACKGROUND: Myeloid cells (MC) have potent immunoregulatory abilities that can be therapeutically useful to treat inflammatory disease. However, the factors which promote regulatory myeloid cell differentiation remain poorly understood. We have previously shown that estriol (E3) induces mature regulatory dendritic cells in vivo. To determine whether additional steroid hormones could induce mature regulatory myeloid cells, we investigated the effects of retinoic acid (RA) on MCs. Retinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation. We hypothesized that the presence of RA during differentiation would promote the formation of mature regulatory myeloid cells (MCregs). METHODS: To determine RA's ability to induce regulatory myeloid cells, we differentiated bone marrow progenitor cells with granulocytic-macrophage colony-stimulating factor (GM-CSF) under the influence of RA. We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells. Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS. CONCLUSION: RA induced mature regulatory myeloid cells that were suppressive and had a CD11b+ CD11c-Ly6C low/intermediate monocyte phenotype. Surprisingly, RA CD11c+ dendritic cells were not suppressive and could contribute to enhanced proliferation. These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

Treatment of experimental autoimmune encephalomyelitis by codelivery of disease associated Peptide and dexamethasone in acetalated dextran microparticles.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system that can cause loss of motor function and is thought to result, in part, from chronic inflammation due to an antigen-specific T cell immune response. Current treatments suppress the immune system without antigen specificity, increasing the risks of cancer, chronic infection, and other long-term side effects. In this study, we show treatment of experimental autoimmune encephalomyelitis (EAE), a model of MS, by coencapsulating the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) with dexamethasone (DXM) into acetalated dextran (Ac-DEX) microparticles (DXM/MOG/MPs) and administering the microparticles subcutaneously. The clinical score of the mice was reduced from 3.4 to 1.6 after 3 injections 3 days apart with the coencapsulated microparticulate formulation (MOG 17.6 µg and DXM 8 µg). This change in clinical score was significantly greater than observed with phosphate-buffered saline (PBS), empty MPs, free DXM and MOG, DXM/MPs, and MOG/MPs. Additionally, treatment with DXM/MOG/MPs significantly inhibited disease-associated cytokine (e.g., IL-17, GM-CSF) expression in splenocytes isolated in treated mice. Here we show a promising approach for the therapeutic treatment of MS using a polymer-based microparticle delivery platform.

miR-29ab1 deficiency identifies a negative feedback loop controlling Th1 bias that is dysregulated in multiple sclerosis.

Th cell programming and function is tightly regulated by complex biological networks to prevent excessive inflammatory responses and autoimmune disease. The importance of microRNAs (miRNAs) in this process is highlighted by the preferential Th1 polarization of Dicer-deficient T cells that lack miRNAs. Using genetic knockouts, we demonstrate that loss of endogenous miR-29, derived from the miR-29ab1 genomic cluster, results in unrestrained T-bet expression and IFN-γ production. miR-29b regulates T-bet and IFN-γ via a direct interaction with the 3' untranslated regions, and IFN-γ itself enhances miR-29b expression, establishing a novel regulatory feedback loop. miR-29b is increased in memory CD4(+) T cells from multiple sclerosis (MS) patients, which may reflect chronic Th1 inflammation. However, miR-29b levels decrease significantly upon T cell activation in MS patients, suggesting that this feedback loop is dysregulated in MS patients and may contribute to chronic inflammation. miR-29 thus serves as a novel regulator of Th1 differentiation, adding to the understanding of T cell-intrinsic regulatory mechanisms that maintain a balance between protective immunity and autoimmunity.

Dimethyl fumarate inhibits dendritic cell maturation via nuclear factor κB (NF-κB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 (MSK1) signaling.

Dimethyl fumarate (DMF) is an effective novel treatment for multiple sclerosis in clinical trials. A reduction of IFN-γ-producing CD4(+) T cells is observed in DMF-treated patients and may contribute to its clinical efficacy. However, the cellular and molecular mechanisms behind this clinical observation are unclear. In this study, we investigated the effects of DMF on dendritic cell (DC) maturation and subsequent DC-mediated T cell responses. We show that DMF inhibits DC maturation by reducing inflammatory cytokine production (IL-12 and IL-6) and the expression of MHC class II, CD80, and CD86. Importantly, this immature DC phenotype generated fewer activated T cells that were characterized by decreased IFN-γ and IL-17 production. Further molecular studies demonstrated that DMF impaired nuclear factor κB (NF-κB) signaling via reduced p65 nuclear translocalization and phosphorylation. NF-κB signaling was further decreased by DMF-mediated suppression of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its downstream kinase mitogen stress-activated kinase 1 (MSK1). MSK1 suppression resulted in decreased p65 phosphorylation at serine 276 and reduced histone phosphorylation at serine 10. As a consequence, DMF appears to reduce p65 transcriptional activity both directly and indirectly by promoting a silent chromatin environment. Finally, treatment of DCs with the MSK1 inhibitor H89 partially mimicked the effects of DMF on the DC signaling pathway and impaired DC maturation. Taken together, these studies indicate that by suppression of both NF-κB and ERK1/2-MSK1 signaling, DMF inhibits maturation of DCs and subsequently Th1 and Th17 cell differentiation.