Lawrence R. Shiow

CD69 acts downstream of interferon-|[alpha]|/|[beta]| to inhibit S1P1 and lymphocyte egress from lymphoid organs

Naive lymphocytes continually enter and exit lymphoid organs in a recirculation process that is essential for immune surveillance. During immune responses, the egress process can be shut down transiently. When this occurs locally it increases lymphocyte numbers in the responding lymphoid organ; when it occurs systemically it can lead to immunosuppression as a result of the depletion of recirculating lymphocytes. Several mediators of the innate immune system are known to cause shutdown, including interferon α/β (IFN-α/β) and tumour necrosis factor, but the mechanism has been unclear. Here we show that treatment with the IFN-α/β inducer polyinosine polycytidylic acid (hereafter ‘poly(I:C)’) inhibited egress by a mechanism that was partly lymphocyte-intrinsic. The transmembrane C-type lectin CD69 was rapidly induced and CD69-/- cells were poorly retained in lymphoid tissues after treatment with poly(I:C) or infection with lymphocytic choriomeningitis virus. Lymphocyte egress requires sphingosine 1-phosphate receptor-1 (S1P1), and IFN-α/β was found to inhibit lymphocyte responsiveness to S1P. By contrast, CD69-/- cells retained S1P1 function after exposure to IFN-α/β. In coexpression experiments, CD69 inhibited S1P1 chemotactic function and led to downmodulation of S1P1. In a reporter assay, S1P1 crosslinking led to co-crosslinking and activation of a CD69–CD3ζ chimaera. CD69 co-immunoprecipitated with S1P1 but not the related receptor, S1P3. These observations indicate that CD69 forms a complex with and negatively regulates S1P1 and that it functions downstream of IFN-α/β, and possibly other activating stimuli, to promote lymphocyte retention in lymphoid organs.

The actin regulator coronin 1A is mutant in a thymic egress–deficient mouse strain and in a patient with severe combined immunodeficiency

Mice carrying the recessive locus for peripheral T cell deficiency (Ptcd) have a block in thymic egress, but the mechanism responsible is undefined. Here we found that Ptcd T cells had an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus showed a point substitution of lysine for glutamic acid at position 26 in the actin regulator coronin 1A that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. The discovery of another coronin 1A mutant during an N-ethyl-N-nitrosourea-mutagenesis screen for T cell–lymphopenic mice prompted us to evaluate a T cell–deficient, B cell–sufficient and natural killer cell–sufficient patient with severe combined immunodeficiency, whom we found had mutations in both CORO1A alleles. Our findings establish a function for coronin 1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation and demonstrate that actin regulation is a biological process defective in human and mouse severe combined immunodeficiency.

CD69 Suppresses Sphingosine 1-Phosophate Receptor-1 (S1P1) Function through Interaction with Membrane Helix 4

Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P1). The activation antigen CD69 associates with and inhibits the function of S1P1, inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P1-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P1 showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P1 in cell lysates, likely reflecting degradation. Unexpectedly, the S1P1-CD69 complex exhibited a much longer half-life for binding of S1P than S1P1 alone. In contrast to wild-type CD69, a non-S1P1 binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P1 and suggest that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1P1 internalization and degradation.

Severe combined immunodeficiency (SCID) and attention deficit hyperactivity disorder (ADHD) associated with a Coronin-1A mutation and a chromosome 16p11.2 deletion.

Defects causing severe combined immunodeficiency (SCID) have been reported in pathways mediating antigen receptor rearrangement, antigen receptor and cytokine signaling, and purine metabolism. Recognizing that the actin regulator Coronin-1A is essential for development of a normal peripheral T cell compartment in mouse models, we identified absence of Coronin-1A in a girl with T-B+NK+ SCID who suffered recurrent infections including severe post-vaccination varicella at age 13 months. Murine Coronin-1A is essential for the release of T cells from the thymus, consistent with the paradoxically detectable thymus in our patient. Molecular analysis revealed a 2 bp deletion in the paternal CORO1A coding sequence paired with a 600 kb de novo deletion encompassing CORO1A on the maternal allele. This genomic region at 16p11.2 is subject to recurrent copy number variations associated with autism spectrum disorders, including attention deficit and hyperactivity, present in our patient. This case highlights the first link between actin cytoskeleton regulation and SCID.

Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer

In colon cancer, mutation of the Wnt repressor APC (encoding adenomatous polyposis coli) leads to a state of aberrant and unrestricted high-activity signaling. However, the relevance of high Wnt tone in non-genetic human disease is unknown. Here we demonstrate that distinct functional states of Wnt activity determine oligodendrocyte precursor cell (OPC) differentiation and myelination. Mouse OPCs with genetic Wnt dysregulation (high tone) express multiple genes in common with colon cancer, including Lef1, Sp5, Ets2, Rnf43 and Dusp4. Surprisingly, we found that OPCs in lesions of hypoxic human neonatal white matter injury upregulated markers of high Wnt activity and lacked expression of APC. We also found that lack of Wnt repressor tone promoted permanent white matter injury after mild hypoxic insult. These findings suggest a state of pathological high-activity Wnt signaling in human disease tissues that lack predisposing genetic mutation.

InlP, a New Virulence Factor with Strong Placental Tropism

ABSTRACT Intrauterine infection is a major detriment for maternal-child health and occurs despite local mechanisms that protect the maternal-fetal interface from a wide variety of pathogens. The bacterial pathogen Listeria monocytogenes causes spontaneous abortion, stillbirth, and preterm labor in humans and serves as a model for placental pathogenesis. Given the unique immunological environment of the maternal-fetal interface, we hypothesized that virulence determinants with placental tropism are required for infection of this tissue. We performed a genomic screen in pregnant guinea pigs that led to the identification of 201 listerial genes important for infection of the placenta but not maternal liver. Among these genes was lmrg1778 (lmo2470), here named inlP, predicted to encode a secreted protein that belongs to the internalin family. InlP is conserved in virulent L. monocytogenes strains but absent in Listeria species that are nonpathogenic for humans. The intracellular life cycle of L. monocytogenes deficient in inlP (ΔinlP) was not impaired. In guinea pigs and mice, InlP increased the placental bacterial burden by a factor of 3 log10 while having only a minor role in other maternal organs. Furthermore, the ΔinlP strain was attenuated in intracellular growth in primary human placental organ cultures and trophoblasts. InlP is a novel virulence factor for listeriosis with a strong tropism for the placenta. This virulence factor represents a tool for the development of new modalities to prevent and treat infection-related pregnancy complications.

Reactive astrocyte COX2-PGE2 production inhibits oligodendrocyte maturation in neonatal white matter injury.

Inflammation is a major risk factor for neonatal white matter injury (NWMI), which is associated with later development of cerebral palsy. Although recent studies have demonstrated maturation arrest of oligodendrocyte progenitor cells (OPCs) in NWMI, the identity of inflammatory mediators with direct effects on OPCs has been unclear. Here, we investigated downstream effects of pro‐inflammatory IL‐1β to induce cyclooxygenase‐2 (COX2) and prostaglandin E2 (PGE2) production in white matter. First, we assessed COX2 expression in human fetal brain and term neonatal brain affected by hypoxic‐ischemic encephalopathy (HIE). In the developing human brain, COX2 was expressed in radial glia, microglia, and endothelial cells. In human term neonatal HIE cases with subcortical WMI, COX2 was strongly induced in reactive astrocytes with “A2” reactivity. Next, we show that OPCs express the EP1 receptor for PGE2, and PGE2 acts directly on OPCs to block maturation in vitro. Pharmacologic blockade with EP1‐specific inhibitors (ONO‐8711, SC‐51089), or genetic deficiency of EP1 attenuated effects of PGE2. In an IL‐1β‐induced model of NWMI, astrocytes also exhibit “A2” reactivity and induce COX2. Furthermore, in vivo inhibition of COX2 with Nimesulide rescues hypomyelination and behavioral impairment. These findings suggest that neonatal white matter astrocytes can develop “A2” reactivity that contributes to OPC maturation arrest in NWMI through induction of COX2‐PGE2 signaling, a pathway that can be targeted for neonatal neuroprotection.