The miR-142 miRNAs: Shaping the naïve immune system.

Immunity in a naïve organism is tightly controlled. Adequate proportions of the many immune cell subsets must be produced to mount efficient responses to eventual challenges. In addition, a functioning immune system is highly dynamic at steady state. Mature immune cells must be positioned properly and/or circulate to facilitate the detection of dangers. They must also be poised to promptly react to unusual encounters, while ignoring innocuous germs and self. Numerous regulatory mechanisms act at the molecular level to generate such an exquisite structure, including miRNA-mediated repression of protein synthesis. Notably, the miRNAs from the miR-142 locus are preferentially expressed in hematopoietic cells. Their importance is underscored by the deeply disturbed immune system seen upon inactivation of the locus in mice. In this review, we explore reported roles for the miR-142 miRNAs in the shaping of immunity in vertebrates, discussing in particular their contributions to the generation, migration and survival of hematopoietic cells.

miR-142 favors naïve B cell residence in peripheral lymph nodes.

B lymphocyte development proceeds through a well-ordered sequence of steps, leading to the formation of a sizeable mature B population recognizing a diversity of antigens. These latter cells are ultimately responsible for the production of antibodies upon immune challenges. The detection of threats to the organism is facilitated by the ability of naïve follicular B cells, the main subset of mature B cells in mice, to circulate between lymphoid tissues in search of their cognate antigens. miRNA-mediated fine-tuning of mRNA stability and translation participates in the optimal expression of genetic programs. This regulatory mechanism has been shown to contribute to B cell biology, although the role of individual miRNAs remains understudied. Here, we selectively inactivated the miR-142 locus in B cells. As a consequence, the mature B compartment was visibly perturbed, in agreement with work in miR-142 knockout mice. However, our strategy allowed us to identify roles for the miR-142 locus in B cell physiology obscured by the complexity of the immune phenotype in the null mutant mice. Thus, these miRNAs are necessary for the proper formation of the pre-B cell compartment during development. More remarkably, naïve follicular B cells demonstrated altered migratory properties upon conditional inactivation of the miR-142 locus. The latter mutant cells expressed reduced levels of the homing molecule CD62L. They also migrated more efficiently towards sphingosine-1-phosphate in vitro and displayed an increased abundance of the sphingosine-1-phosphate receptor 1, compatible with improved lymphocyte egress in vivo. In line with these observations, the ablation of the miR-142 locus in B cells caused a paucity of B cells in the lymph nodes. Mutant B cell accumulation in the latter tissues was also compromised upon transfer into a wild-type environment. These changes coincided with suboptimal levels of FOXO1, a positive regulator of CD62L transcription, in mutant B cells. Overall, our findings indicate contributions for the miR-142 locus in various aspects of the B cell life cycle. Notably, this locus appears to favor the establishment of the migratory behavior required for naïve follicular B cell patrolling activity.

A role for the nuclear receptor NR2F6 in peritoneal B cell homeostasis.

B cells are key mediators of humoral immunity. Mature B cells fall into various sub-classes that can be separated by their ontogeny, expression of cell surface markers, anatomical location, and function. B1 subsets play important roles in natural immunity and constitute the majority of B cells in newborns. In the adult, B1 cells predominate in the pleural and peritoneal cavities, while the mature B2 follicular subset makes up the major fraction of B cells in lymphoid tissue, although important subsets of antibody-secreting B1 cells are also present at these sites. B1 cells are the main producers of natural IgM but can also contribute to elimination of some pathogens, while B2 cells primarily mediate response to foreign antigens. The differential molecular underpinning of the B1 and B2 subsets remains incompletely understood. Here we demonstrate that germline-deficiency of the orphan nuclear receptor NR2F6 causes a partial loss of B1b and B2 B cells in the peritoneum while leaving peritoneal B1a cells unaltered. A competitive bone marrow chimera in Nr2f6+/+ host mice produced similar numbers of Nr2f6+/+ and Nr2f6-/- peritoneal B1b and B2 cells. The proliferation of Nr2f6-/- peritoneal B cells was not altered, while the migration marker CXCR5 was reduced on all subsets but Beta7-integrin was reduced only on peritoneal B1b and B2 cells. Similarly, B1b and B2 but not B1a cells, exhibited significantly reduced survival.

Addressing the role of PKD3 in the T cell compartment with knockout mice.

BACKGROUND: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. METHODS: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. RESULTS: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. CONCLUSION: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127-KLRG1+) or memory precursors cells (MPECs, CD127+KLRG1-) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8+ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6-/- OT-I T-cells showed that the augmented memory formation is CD8+ T-cell intrinsic. Although the relative difference between the Nr2f6+/+ and Nr2f6-/- OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8+ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8+ T cells in vivo.

Bacterial Infection with Listeria monocytogenes in Mice and Subsequent Analysis of Antigen-Specific CD8 T Cell Responses.

Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium Listeria monocytogenes serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon Listeria monocytogenes (LmOVA) infection ( Jakic et al., 2021 ). The current protocol details how to cultivate ovalbumin-expressing LmOVA, infect naïve C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells in vivo following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naïve OT-I CD8 T cells into congenic hosts before LmOVA infection. Graphic abstract: Figure 1.Experimental workflow depicting the steps for infection of mice with Listeria and subsequent analysis of antigen-specific CD8 memory responses. Bacteria (ovalbumin expressing Listeria monocytogenes) are thawed and grown on lysogeny broth (LB) plates overnight (ON). A single colony is picked and grown in LB medium ON. Bacteria from the exponential growth phase are then injected into a C57BL/6 mouse via tail vein injection. Colony forming units (CFU) of the bacteria can be detected in the spleen on day 3 post injection. Antigen-specific CD8 T cell immune response can be investigated during the acute phase (d3 after infection), during the peak of the adaptive immune response (d7), the clearance phase (d26), or the memory phase (d70) by flow cytometry. Created with BioRender.com.

Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+.

Regulation of the germinal center response by nuclear receptors and implications for autoimmune diseases.

The immune system plays an essential role in protecting the host from infectious diseases and cancer. Notably, B and T lymphocytes from the adaptive arm of the immune system can co-operate to form long-lived antibody responses and are therefore the main target in vaccination approaches. Nevertheless, protective immune responses must be tightly regulated to avoid hyper-responsiveness and responses against self that can result in autoimmunity. Nuclear receptors (NRs) are perfectly adapted to rapidly alter transcriptional cellular responses to altered environmental settings. Their functional role is associated with both immune deficiencies and autoimmunity. Despite extensive linking of nuclear receptor function with specific CD4 T helper subsets, research on the functional roles and mechanisms of specific NRs in CD4 follicular T helper cells (Tfh) and germinal center (GC) B cells during the germinal center reaction is just emerging. We review recent advances in our understanding of NR regulation in specific cell types of the GC response and discuss their implications for autoimmune diseases such as systemic lupus erythematosus (SLE).

Orphan Nuclear Receptor NR2F6 Suppresses T Follicular Helper Cell Accumulation through Regulation of IL-21.

CD4 T follicular helper (Tfh) cells are specialized in helping B cells during the germinal center (GC) reaction and ultimately promote long-term humoral immunity. Here we report that loss of the nuclear orphan receptor NR2F6 causes enhanced survival and accumulation of Tfh cells, GC B cells, and plasma cells (PCs) following T cell-dependent immunization. Nr2f6-deficient CD4 T cell dysfunction is the primary cause of cell accumulation. Cytokine expression in Nr2f6-deficient Tfh cells is dysregulated, and Il21 expression is enhanced. Mechanistically, NR2F6 binds directly to the interleukin 21 (IL-21) promoter and a conserved noncoding sequence (CNS) near the Il21 gene in resting CD4+ T cells. During Tfh cell differentiation, this direct NR2F6 DNA interaction is abolished. Enhanced Tfh cell accumulation in Nr2f6-deficient mice can be reverted by blocking IL-21R signaling. Thus, NR2F6 is a critical negative regulator of IL-21 cytokine production in Tfh cells and prevents excessive Tfh cell accumulation.

Nuclear orphan receptor NR2F6 as a safeguard against experimental murine colitis.

OBJECTIVE: Nuclear receptors are known to regulate both immune and barrier functions in the GI tract. The nuclear orphan receptor NR2F6 has been shown to suppress the expression of proinflammatory cytokines in T lymphocytes. NR2F6 gene expression is reduced in patients with IBS or UC, but its functional role and tissue dependency in healthy and inflamed gut have not yet been investigated. DESIGN: Intestinal inflammation was induced in wild-type, Nr2f6-deficient, Rag1-deficient or bone marrow-reconstituted mice by administration of chemical (dextran sodium sulfate (DSS)) and immunogenic (T cell transfer) triggers. Disease phenotypes were investigated by survival, body weight, colon length and analysis of immune cell infiltrates. Additionally, histology, intestinal permeability, tight junction proteins, bacterial fluorescence in situ hybridisation, apoptosis, cell proliferation and mucus production were investigated. RESULTS: Nr2f6-deficient mice were highly susceptible to DSS-induced colitis characterised by enhanced weight loss, increased colonic tissue destruction and immune cell infiltration together with enhanced intestinal permeability and reduced Muc2 expression. T cell transfer colitis and bone marrow reconstitution experiments demonstrated that disease susceptibility was not dependent on the expression of Nr2f6 in the immune compartment but on the protective role of NR2F6 in the intestinal epithelium. Mechanistically, we show that NR2F6 binds to a consensus sequence at -2 kb of the Muc2 promoter and transactivates Muc2 expression. Loss of NR2F6 alters intestinal permeability and results in spontaneous late-onset colitis in Nr2f6-deficient mice. CONCLUSION: We have for the first time identified a fundamental and non-redundant role of NR2F6 in protecting gut barrier homeostasis.