Functional plasticity shapes neutrophil response to Leishmania major infection in susceptible and resistant strains of mice.

Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice.

Kupffer cell and recruited macrophage heterogeneity orchestrate granuloma maturation and hepatic immunity in visceral leishmaniasis.

In murine models of visceral leishmaniasis (VL), parasitization of resident Kupffer cells (resKCs) is responsible for early growth of Leishmania infantum in the liver, which leads to granuloma formation and eventual parasite control. We employed the chronic VL model, and revealed an open niche established by KCs death and their migration outside of the sinusoids, resulting in their gradual replacement by monocyte-derived KCs (moKCs). While early granulomas were composed of resKCs, late granulomas were found outside of the sinusoids and contained resKC-derived macrophages, and monocyte-derived macrophages (momacs). ResKCs and moKCs within the sinusoids were identified as homeostatic/regulatory cells, while resKC-derived macrophages and momacs within late granulomas were pro-inflammatory. Despite the infection being largely confined to the resKC-derived macrophages, in the absence of monocyte recruitment, parasite control was strongly compromised. Macrophage heterogeneity, involving migration and reprogramming of resKCs, along with recruitment of monocyte-derived cells, is a hallmark of granuloma maturation and hepatic immunity in VL.

Single-cell transcriptional analysis of murine norovirus infection in a human intestinal cell line.

Noroviruses are a major agent of acute gastroenteritis in humans, but host cell requirements for efficient replication in vitro have not been established. We engineered a human intestinal cell line (designated mCD300lf-hCaco2) expressing the murine norovirus (MNV) receptor, mouse CD300lf to become fully permissive for MNV replication. To explore the replicative machinery and host response of these cells, we performed a single-cell RNA sequencing (scRNA-seq) transcriptomics analysis of an MNV infection over time. Marked similarities were observed between certain global features of MNV infection in human cells compared to those previously reported in mouse cells by whole population transcriptomics such as downregulation of ribosome biogenesis, mitochondrial dysfunction, and cell cycle preference for G1. Our scRNA-seq analysis allowed further resolution of an infected cell population into distinct clusters with varying levels of viral RNA and interferon-stimulated gene ISG15 transcripts. Cells with high viral replication displayed downregulated ribosomal protein small (RPS) and large (RPL) genes and mitochondrial complexes I, III, IV, and V genes during exponential viral propagation. Ferritin subunit genes FTL and FTH1 were also downregulated during active MNV replication, suggesting that inhibition of iron metabolism may increase replication efficiency. Consistent with this, transcriptional activation of these genes with ferric ammonium citrate and overexpression of FTL lowered virus yields. Comparative studies of cells that support varying levels of norovirus replication efficiency, as determined by scRNA-seq may lead to improved human cell-based culture systems and effective viral interventions.IMPORTANCEHuman noroviruses cause acute gastroenteritis in all age groups. Vaccines and antiviral drugs are not yet available, in part, because it is difficult to propagate the viruses causing human disease in standard laboratory cell culture systems. In contrast, a norovirus found in mice [murine norovirus (MNV)] replicates efficiently in murine-based cell culture and has served as a model system. In this study, we established a new human intestinal cell line that was genetically modified to express the murine norovirus receptor so that the human cells became permissive to murine norovirus infection. We then defined the host response to MNV infection in the engineered human cell line at a single-cell resolution and identified cellular genes associated with the highest levels of MNV replication. This study may lead to the improvement of the current human norovirus cell culture systems and help to identify norovirus-host interactions that could be targeted for antiviral drugs.

PARP15 is a Susceptibility Locus for Clarkson Disease (Monoclonal Gammopathy-Associated Systemic Capillary Leak Syndrome).

BACKGROUND: Vascular leakage is a deadly complication of severe infections, ranging from bacterial sepsis to malaria. Worldwide, septicemia is among the top 10 causes of lethality because of the shock and multiorgan dysfunction that arise from the host vascular response. In the monoclonal gammopathy-associated capillary leak syndrome (MG-CLS), even otherwise mundane infections induce recurrent septic-like episodes of profound microvascular hyperpermeability and shock. There are no defined genetic risk factors for MG-CLS or effective treatments for acute crises. METHODS: We characterized predicted loss-of-function mutations in PARP15 (poly[ADP-ribose] polymerase 15), a protein of unknown function that is absent in mice, in patients with MG-CLS. We analyzed barrier function in PARP15-deficient vascular endothelial cells and vascular leakage in mice engineered to express WT or loss-of-function variant human PARP15. RESULTS: We discovered several loss-of-function PARP15 variants associated with MG-CLS. These mutations severely reduced PARP15 enzymatic function. The presence of the most frequently detected variant (G628R) correlated with clinical markers of severe vascular leakage. In human microvascular endothelial cells, PARP15 suppressed cytokine-induced barrier disruption by ADP-ribosylating the scaffold protein JIP3 (c-Jun N-terminal kinase-interacting protein 3) and inhibiting p38 MAP kinase activation. Mice expressing enzymatically inactive human PARP15(G628R) were significantly more prone to inflammation-associated vascular leakage than mice expressing WT PARP15 in a p38-dependent fashion. CONCLUSIONS: PARP15 represents a previously unrecognized genetic susceptibility factor for MG-CLS. PARP15-mediated ADP ribosylation is an essential and genetically determined mechanism of the human vascular response to inflammation.

Germline mutations in a G protein identify signaling cross-talk in T cells.

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes Gαi2, a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating Gαi2 mutations had clinical presentations that included impaired immunity. Mutant Gαi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant Gαi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.

A deep intronic splice-altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

TRPA1 Influences Staphylococcus aureus Skin Infection in Mice and Associates with HIF-1a and MAPK Pathway Modulation.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major public health burden. Emerging antibiotic resistance has heightened the need for new treatment approaches for MRSA infection such as developing novel antimicrobial agents and enhancing the host's defense response. The thermo-ion channels Transient Receptor Potential (TRP-) A1 and V1 have been identified as modulators of S. aureus quorum sensing in cell culture models. However, their effects on in vivo infection control are unknown. In this study, we investigated the therapeutic effect of natural TRP ion channel inhibitors on MRSA skin infection in mice. While deletion of TRPV1 did not affect lesion size or inflammatory markers, TRPA1-/- mice demonstrated significantly reduced infection severity and abscess size. Treatment with natural inhibitors of TRPA1 with or without blockade of TRPV1 also reduced abscess size. Tissue transcriptomic data coupled with immunohistochemistry revealed that TRPA1 inhibition impacted heat shock protein expression (HSP), modulated the HIF-1a and MAPK pathways, and reduced IL4 expression. Additionally, metabolomics data showed an impact on purine and glycosaminoglycan pathways. Multi-omic integration of transcriptomic and metabolic data revealed that diacylglycerol metabolism was the likely bridge between metabolic and immunological impacts. Our findings suggest that TRPA1 antagonism could provide a promising and cost-effective therapeutic approach for reducing the severity of MRSA infection, and presents a novel underlying molecular mechanism.

Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation.

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

Optimal CXCR5 Expression during Tfh Maturation Involves the Bhlhe40-Pou2af1 Axis.

The pair of transcription factors Bcl6-Blimp1 is well-known for follicular T helper (Tfh) cell fate determination, however, the mechanism(s) for Bcl6-independent regulation of CXCR5 during Tfh migration into germinal center (GC) is still unclear. In this study, we uncovered another pair of transcription factors, Bhlhe40-Pou2af1, that regulates CXCR5 expression. Pou2af1 was specifically expressed in Tfh cells whereas Bhlhe40 expression was found high in non-Tfh cells. Pou2af1 promoted Tfh formation and migration into GC by upregulating CXCR5 but not Bcl6, while Bhlhe40 repressed this process by inhibiting Pou2af1 expression. RNA-Seq analysis of antigen-specific Tfh cells generated in vivo confirmed the role of Bhlhe40-Pou2af1 axis in regulating optimal CXCR5 expression. Thus, the regulation of CXCR5 expression and migration of Tfh cells into GC involves a transcriptional regulatory circuit consisting of Bhlhe40 and Pou2af1, which operates independent of the Bcl6-Blimp1 circuit that determines the Tfh cell fate.

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo . Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statement: When cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo . Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection.

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-ß and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/ß levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-ß mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-ß responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

MAdCAM-1 co-stimulation combined with retinoic acid and TGF-ß induces blood CD8+ T cells to adopt a gut CD101+ TRM phenotype.

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue-protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that mucosal vascular addressin cell adhesion molecule 1, a ligand for the gut-homing receptor α4ß7 integrin, in the presence of retinoic acid and transforming growth factor-ß (TGF-ß) provides a co-stimulatory signal that induces blood cluster of differentiation (CD8+ T cells to adopt a TRM-like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell-surface markers, along with CD101. They also express C-C motif chemokine receptors 5 (CCR5) , C-C motif chemokine receptors 9 (CCR9), and α4ß7, three receptors associated with gut homing. A subset also expresses E-cadherin, a ligand for αEß7. Fluorescent lifetime imaging indicated an αEß7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into resident memory T cells and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue-specific immunotherapies.

MATRIN3 deficiency triggers autoinflammation via cGAS-STING activation.

Interferon-stimulated genes (ISGs) comprise a program of immune effectors important for host immune defense. When uncontrolled, ISGs play a central role in interferonopathies and other inflammatory diseases. The mechanisms responsible for turning on ISGs are not completely known. By investigating MATRIN3 (MATR3), a nuclear RNA-binding protein mutated in familial ALS, we found that perturbing MATR3 results in elevated expression of ISGs. Using an integrative approach, we elucidate a pathway that leads to activation of cGAS-STING. This outlines a plausible mechanism for pathogenesis in a subset of ALS, and suggests new diagnostic and therapeutic approaches for this fatal disease.

Topical Steroid Withdrawal is a Targetable Excess of Mitochondrial NAD.

Background: Topical corticosteroids (TCS) are first-line therapies for numerous skin conditions. Topical Steroid Withdrawal (TSW) is a controversial diagnosis advocated by patients with prolonged TCS exposure who report severe systemic reactions upon treatment cessation. However, to date there have been no systematic clinical or mechanistic studies to distinguish TSW from other eczematous disorders. Methods: A re-analysis of a previous survey with eczematous skin disease was performed to evaluate potential TSW distinguishing symptoms. We subsequently conducted a pilot study of 16 patients fitting the proposed diagnostic criteria. We then performed: tissue metabolomics, transcriptomics, and immunostaining on skin biopsies; serum metabolomics and cytokine assessments; shotgun metagenomics on microbiome skin swabs; genome sequencing; followed by functional, mechanistic studies using human skin cell lines and mice. Results: Clinically distinct TSW symptoms included burning, flushing, and thermodysregulation. Metabolomics and transcriptomics both implicated elevated NAD+ oxidation stemming from increased expression of mitochondrial complex I and conversion of tryptophan into kynurenine metabolites. These abnormalities were induced by glucocorticoid exposure both in vitro and in a cohort of healthy controls (N=19) exposed to TCS. Targeting complex I via either metformin or the herbal compound berberine improved outcomes in both cell culture and in an open-label case series for patients with TSW. Conclusion: Taken together, our results suggest that TSW has a distinct dermatopathology. While future studies are needed to validate these results in larger cohorts, this work provides the first mechanistic evaluation into TSW pathology, and offers insights into clinical identification, pharmacogenomic candidates, and directed therapeutic strategies.

Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway.

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

The V2 domain of HIV gp120 mimics an interaction between CD4 and integrin ⍺4ß7.

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4ß7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4ß7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4ß7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G ß-strand of CD4 D2, KIDIV, that binds directly to ⍺4ß7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4ß7 binding. The accessory ⍺4ß7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4ß7. As such, the interactions of gp120 with both CD4 and ⍺4ß7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4ß7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors.

Antimony Compounds Associate with Atopic Dermatitis and Influence Models of Itch and Dysbiosis.

Compared to the myriad of known triggers for rhinitis and asthma, environmental exposure research for atopic dermatitis (AD) is not well established. We recently reported that an untargeted search of U.S. Environmental Protection Agency (EPA) databases versus AD rates by United States (U.S.) postal codes revealed that isocyanates, such as toluene diisocyanate (TDI), are the pollutant class with the strongest spatiotemporal and epidemiologic association with AD. We further demonstrated that (di)isocyanates disrupt ceramide-family lipid production in commensal bacteria and activate the thermo-itch host receptor TRPA1. In this report, we reanalyzed regions of the U.S. with low levels of diisocyanate pollution to assess if a different chemical class may contribute. We identified antimony compounds as the top associated pollutant in such regions. Exposure to antimony compounds would be expected from brake dust in high-traffic areas, smelting plants, bottled water, and dust from aerosolized soil. Like TDI, antimony inhibited ceramide-family lipid production in Roseomonas mucosa and activated TRPA1 in human neurons. While further epidemiologic research will be needed to directly evaluate antimony exposure with surrounding AD prevalence and severity, these data suggest that compounds which are epidemiologically associated with AD, inhibit commensal lipid production, and activate TRPA1 may be causally related to AD pathogenesis.

Specific regulation of mechanical nociception by Gß5 involves GABA-B receptors.

Mechanical, thermal, and chemical pain sensation is conveyed by primary nociceptors, a subset of sensory afferent neurons. The intracellular regulation of the primary nociceptive signal is an area of active study. We report here the discovery of a Gß5-dependent regulatory pathway within mechanical nociceptors that restrains antinociceptive input from metabotropic GABA-B receptors. In mice with conditional knockout (cKO) of the gene that encodes Gß5 (Gnb5) targeted to peripheral sensory neurons, we demonstrate the impairment of mechanical, thermal, and chemical nociception. We further report the specific loss of mechanical nociception in Rgs7-Cre+/- Gnb5fl/fl mice but not in Rgs9-Cre+/- Gnb5fl/fl mice, suggesting that Gß5 might specifically regulate mechanical pain in regulator of G protein signaling 7-positive (Rgs7+) cells. Additionally, Gß5-dependent and Rgs7-associated mechanical nociception is dependent upon GABA-B receptor signaling since both were abolished by treatment with a GABA-B receptor antagonist and since cKO of Gß5 from sensory cells or from Rgs7+ cells potentiated the analgesic effects of GABA-B agonists. Following activation by the G protein-coupled receptor Mrgprd agonist ß-alanine, enhanced sensitivity to inhibition by baclofen was observed in primary cultures of Rgs7+ sensory neurons harvested from Rgs7-Cre+/- Gnb5fl/fl mice. Taken together, these results suggest that the targeted inhibition of Gß5 function in Rgs7+ sensory neurons might provide specific relief for mechanical allodynia, including that contributing to chronic neuropathic pain, without reliance on exogenous opioids.