Mice Expressing Cosegregating Single Nucleotide Polymorphisms (D298G and N397I) in TLR4 Have Enhanced Responses to House Dust Mite Allergen.

Asthma is a common and ubiquitous chronic respiratory disease that is associated with airway inflammation and hyperreactivity resulting in airway obstruction. It is now accepted that asthma is controlled by a combination of host genetics and environment in a rather complex fashion; however, the link between sensing of the environment and development and exacerbation of allergic lung inflammation is unclear. Human populations expressing cosegregating D299G and T399I polymorphisms in the TLR4 gene are associated with a decreased risk for asthma in adults along with hyporesponsiveness to inhaled LPS, the TLR4 ligand. However, these data do not account for other human genetic or environmental factors. Using a novel mouse strain that expresses homologous human TLR4 polymorphisms (TLR4-single nucleotide polymorphism [SNP]), we directly tested the effect of these TLR4 polymorphisms on in vivo responses to allergens using two models of induction. We report that intact TLR4 is required for allergic inflammation when using the OVA and LPS model of induction, as cellular and pathological benchmarks were diminished in both TLR4-SNP and TLR4-deficent mice. However, in the more clinically relevant model using house dust mite extract for induction, responses were enhanced in the TLR4-SNP mice, as evidenced by greater levels of eosinophilic inflammation, Th2 cytokine production, and house dust mite-specific IgG1 production compared with wild-type mice; however, mucus production and airway hyperreactivity were not affected. These results suggest that the TLR4 polymorphic variants (genes) interact differently with the allergic stimulation (environment).

Identifying Function Determining Residues in Neuroimmune Semaphorin 4A.

Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.

Semaphorin 4A Stabilizes Human Regulatory T Cell Phenotype via Plexin B1.

We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T (Treg) cell numbers in vivo; however, the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T cell lines (HuT78 and HuT102), human PBMCs, and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T effector-like phenotype (CD4+CD25lowFoxp3-), whereas HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. HuT102 cells expressed Sema4A counter receptor Plexin B1, whereas HuT78 cells were Sema4A+. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both NRP-1 and -2. However, NRP-1 and Sema4A were detected on CD3negativeCD4intermediate human monocytes. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMCs and CD4+ T cells, which were NRP-1negative but PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before recombinant Sema4A addition significantly decreased Treg cell numbers as compared with cultures with recombinant Sema4A alone. Sema4A was as effective as TGF-ß in inducible Treg cell induction from CD4+CD25depleted cells but did not enhance Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases. ImmunoHorizons, 2019, 3: 71-87.

Oxaliplatin disrupts pathological features of glioma cells and associated macrophages independent of apoptosis induction.

INTRODUCTION: Emerging evidence suggests that effective treatment of glioblastoma (GBM), the most common and deadly form of adult primary brain cancer, will likely require concurrent treatment of multiple aspects of tumor pathobiology to overcome tumor heterogeneity and the complex tumor-supporting microenvironment. Recent studies in non-central nervous system (CNS) tumor cells have demonstrated that oxaliplatin (OXA) can induce multi-faceted anti-tumor effects, in particular at drug concentrations below those required to induce apoptosis. These findings motivated re-investigation of OXA for the treatment of GBM. METHODS: The effects of OXA on murine KR158 and GL261 glioma cells including cell growth, cell death, inhibition of signal transducer and activator of transcription (STAT) activity, O-6-methylguanine-DNA methyltransferase (MGMT) expression, and immunogenic cell death (ICD) initiation, were evaluated by cytotoxicity assays, Western blot analysis, STAT3-luciferase reporter assays, qRT-PCR assays, and flow cytometry. Chemical inhibitors of endoplasmic reticulum (ER) stress were used to investigate the contribution of this cell damage response to the observed OXA effects. The effect of OXA on bone marrow-derived macrophages (BMDM) exposed to glioma conditioned media (GCM) was also analyzed by Western blot analysis. RESULTS: We identified the OXA concentration threshold for induction of apoptosis and from this determined the drug dose and treatment period for sub-cytotoxic treatments of glioma cells. Under these experimental conditions, OXA reduced STAT3 activity, reduced MGMT levels and increased temozolomide sensitivity. In addition, there was evidence of immunogenic cell death (elevated EIF2α phosphorylation and calreticulin exposure) following prolonged OXA treatment. Notably, inhibition of ER stress reversed the OXA-mediated inhibition of STAT3 activity and MGMT expression in the tumor cells. In BMDMs exposed to GCM, OXA also reduced levels of phosphorylated STAT3 and decreased expression of Arginase 1, an enzyme known to contribute to pro-tumor functions in the tumor-immune environment. CONCLUSIONS: OXA can induce notable multi-faceted biological effects in glioma cells and BMDMs at relatively low drug concentrations. These findings may have significant therapeutic relevance against GBM and warrant further investigation.

The adaptor protein insulin receptor substrate 2 inhibits alternative macrophage activation and allergic lung inflammation.

Insulin receptor substrate 2 (IRS2) is an adaptor protein that becomes tyrosine-phosphorylated in response to the cytokines interleukin-4 (IL-4) and IL-13, which results in activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. IL-4 and IL-13 contribute to allergic lung inflammation. To examine the role of IRS2 in allergic disease, we evaluated the responses of IRS2-deficient (IRS2(-/-)) mice. Unexpectedly, loss of IRS2 resulted in a substantial increase in the expression of a subset of genes associated with the generation of alternatively activated macrophages (AAMs) in response to IL-4 or IL-13 in vitro. AAMs secrete factors that enhance allergic responses and promote airway remodeling. Moreover, compared to IRS2(+/+) mice, IRS2(+/-) and IRS2(-/-) mice developed enhanced pulmonary inflammation, accumulated eosinophils and AAMs, and exhibited airway and vascular remodeling upon allergen stimulation, responses that partially depended on macrophage-intrinsic IRS2 signaling. Both in unstimulated and IL-4-stimulated macrophages, lack of IRS2 enhanced phosphorylation of Akt and ribosomal S6 protein. Thus, we identified a critical inhibitory loop downstream of IRS2, demonstrating an unanticipated and previously unrecognized role for IRS2 in suppressing allergic lung inflammation and remodeling.

Absence of the common gamma chain (γ(c)), a critical component of the Type I IL-4 receptor, increases the severity of allergic lung inflammation.

The T(H)2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates T(H)2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling T(H)2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γ(c)) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4⁺ OT-II T cells were adoptively transferred into RAG2⁻/⁻ and γ(c)⁻/⁻ mice and allergic lung disease was induced. Both γ(c)⁻/⁻ and γcxRAG2⁻/⁻ mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2⁻/⁻ mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γ(c)⁻/⁻ mice. Absence of γc in macrophages, however, resulted in reduced YM1 expression. We observed higher T(H)2 cytokine levels in the BAL and an altered DC phenotype in the γ(c)⁻/⁻ recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γ(c)-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of T(H)2 effectors. However, the Type I R regulates AAM protein expression in macrophages.

The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.

Previously, we demonstrated that the γC subunit of type I IL-4 receptor was required for robust tyrosine phosphorylation of the downstream adapter protein, IRS-2, correlating with the expression of genes (ArgI, Retnla, and Chi3l3) characteristic of alternatively activated macrophages. We located an I4R-like motif (IRS-2 docking sequence) in the γC cytoplasmic domain but not in the IL-13Rα1. Thus, we predicted that the γC tail directed enhanced IRS-2 phosphorylation. To test this, IL-4 signaling responses were examined in a mutant of the key I4R motif tyrosine residue (Y325F) and different γC truncation mutants (γ285, γ308, γ318, γ323, and γFULL LENGTH (FL)) co-expressed in L-cells or CHO cells with wild-type (WT) IL-4Rα. Surprisingly, IRS-1 phosphorylation was not diminished in Y325F L-cell mutants suggesting Tyr-325 was not required for the robust insulin receptor substrate response. IRS-2, STAT6, and JAK3 phosphorylation was observed in CHO cells expressing γ323 and γFL but not in γ318 and γ285 mutants. In addition, when CHO cells expressed γ318, γ323, or γFL with IL-2Rß, IL-2 induced phospho-STAT5 only in the γ323 and γFL clones. Our data suggest that a smaller (5 amino acid) interval than previously determined is necessary for JAK3 activation/γC-mediated signaling in response to IL-4 and IL-2. Chimeric receptor chains of the γC tail fused to the IL-13Rα1 extracellular and transmembrane domain did not elicit robust IRS-2 phosphorylation in response to IL-13 suggesting that the extracellular/transmembrane domains of the IL-4/IL-13 receptor, not the cytoplasmic domains, control signaling efficiency. Understanding this pathway fully will lead to rational drug design for allergic disease.

NF-κB signaling participates in both RANKL- and IL-4-induced macrophage fusion: receptor cross-talk leads to alterations in NF-κB pathways.

NF-κB activation is essential for receptor activator for NF-κB ligand (RANKL)-induced osteoclast formation. IL-4 is known to inhibit the RANKL-induced osteoclast differentiation while at the same time promoting macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 inhibition of osteoclastogenesis is mediated by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block proximal, canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in nfκb1(-/-) bone marrow-derived macrophages (BMM), the formation of both multinucleated osteoclast and MNG induced by RANKL or IL-4, respectively, was impaired. This suggests that NF-κB signaling also plays an important role in IL-4-induced macrophage fusion. Indeed, we found that the RANKL-induced and IL-4-induced macrophage fusion were both inhibited by the NF-κB inhibitors IκB kinase 2 inhibitor and NF-κB essential modulator inhibitory peptide. Furthermore, overexpression of p50, p65, p52, and RelB individually in nfκb1(-/-) or nfκb1(+/+) BMM enhanced both giant osteoclast and MNG formation. Interestingly, knockdown of nfκb2 in wild-type BMM dramatically enhanced both osteoclast and MNG formation. In addition, both RANKL- and IL-4-induced macrophage fusion were impaired in NF-κB-inducing kinase(-/-) BMM. These results suggest IL-4 influences NF-κB pathways by increasing p105/p50 and suppressing RANKL-induced p52 translocation and that NF-κB pathways participate in both RANKL- and IL-4-induced giant cell formation.

Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.

Although interleukin-4 (IL-4) and IL-13 participate in allergic inflammation and share a receptor subunit (IL-4Ralpha), they have different functions. We compared cells expressing type I and II IL-4Rs with cells expressing only type II receptors for their responsiveness to these cytokines. IL-4 induced highly efficient, gammaC-dependent tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2), whereas IL-13 was less effective, even when phosphorylation of signal transducer and activator of transcription 6 (STAT6) was maximal. Only type I receptor, gammaC-dependent signaling induced efficient association of IRS-2 with the p85 subunit of phosphoinositide 3-kinase or the adaptor protein growth factor receptor-bound protein 2. In addition, IL-4 signaling through type I IL-4Rs induced more robust expression of a subset of genes associated with alternatively activated macrophages than did IL-13. Thus, IL-4 activates signaling pathways through type I IL-4Rs qualitatively differently from IL-13, which cooperate to induce optimal gene expression.

One new bufadienolide from Chinese drug "Chan'Su".

A new bufadienolide named 16beta-acetoxy-bufarenogin (1), together with six known bufadienolides, namely, 11alpha,12beta-dihydroxy-bufalin (2), bufotalin (3), hellebrigenin (4), desacetylbufotalin (5), gamabufotalin (6), and resibufagin (7) were isolated from Chan'Su. Of these, 2 was a new natural product. Their structures were elucidated by spectral methods. The cytotoxic activities in vitro of these compounds have been assayed against HeLa cell line. They all showed strong cytotoxic activities.

Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4/13 system.

Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R alpha/gamma(c)/IL-4) and type II (IL-4R alpha/IL-13R alpha1/IL-4, IL-4R alpha/IL-13R alpha1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for gamma(c)'s ability to recognize six different gamma(c)-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R alpha1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

IL-4 protects the B-cell lymphoma cell line CH31 from anti-IgM-induced growth arrest and apoptosis: contribution of the PI-3 kinase/AKT pathway.

Interleukin-4 (IL-4) promotes lymphocyte survival and protects primary lymphomas from apoptosis. Previous studies reported differential requirements for the signal transducer and activator of transcription 6 (STAT6) and IRS2/phosphatidylinositol 3 kinase (PI-3K) signaling pathways in mediating the IL-4-induced protection from Fas-mediated apoptosis. In this study, we characterized IL-4-activated signals that suppress anti-IgM-mediated apoptosis and growth arrest of CH31, a model B-cell lymphoma line. In CH31, anti-IgM treatment leads to the loss of mitochondrial membrane potential, phospho-Akt, phospho-CDK2, and c-myc protein. These losses are followed by massive induction of p27(Kip1) protein expression, cell cycle arrest, and apoptosis. Strikingly, IL-4 treatment prevented or reversed these changes. Furthermore, IL-4 suppressed the activation of caspases 9 and 3, and, in contrast to previous reports, induced the phosphorylation (deactivation) of BAD. IL-4 treatment also induced expression of BclxL, a STAT6-dependent gene. Pharmacologic inhibitors and dominant inhibitory forms of PI-3K and Akt abrogated the anti-apoptotic function of IL-4. These results suggest that the IL-4 receptor activates several signaling pathways, with the Akt pathway playing a major role in suppression of the apoptotic program activated by anti-IgM.

A new sesquiterpene lactone with sulfonic acid group from Saussurea lappa.

A new sesquiterpene lactone with an unusual sulfonic acid group, 13-sulfo-dihydrodehydrocostus lactone (1), was isolated from the roots of Saussurea lappa C. (Compositae), together with a known lignan (2). The structure of 1 was characterized on the basis of spectral evidence including 2DNMR studies. Compound 2 was obtained from this plant for the first time.

Microbial transformation of dextromethorphan by Cunninghamella blakesleeana AS 3.153.

The capability of Cunninghamella blakesleeana AS 3.153 to transform CYP2D6 probe drug dextromethorphan was investigated. Metabolites produced by strain AS 3.153 were detected by liquid chromatography-tandem mass spectrometry (LC-MS(n)) and the metabolite dextrorphan was identified by reference to confirm its structure. The yield of dextrorphan produced by C. blakesleeana AS 3.153 was over 90%. Quinidine, a CYP2D6 selective inhibitor, was applied to investigate its effect on biotransformation. The concentration of quinidine was 4-folds higher than that of dextromethorphan and the yield of dextrorphan was reduced by 84%, which proved there was drug metabolism enzyme similar to CYP2D6 in C. blakesleeana AS 3.153. It is concluded that C. blakesleeana AS 3.153 can be used as the suitable model strain in vitro to mimic human CYP2D6 metabolism.

Biotransformation of cinobufagin by Cunninghamella elegans.

Cunninghamella elegans has been employed for the biotransformation of cinobufagin to afford 5 metabolites. The structures of the transformation products have been characterized as 12alpha-hydroxybufagin, 11alpha-hydroxybufagin, 12beta-hydroxy-desacetylcinobufagin, 3-oxo-12alpha-hydroxybufagin and 12beta-hydroxybufagin. Products 12alpha-hydroxybufagin and 11alpha-hydroxybufagin are new compounds. In vitro both the biotransformation products and cinobufagin all showed cytotoxic activities against HeLa cells.

Two new sesquiterpene lactones with the sulfonic acid group from Saussurea lappa.

Two new sesquiterpene lactones with the unusual sulfonic acid group, 13-sulfo-dihydrosantamarine (1) and 13-sulfo-dihydroreynosin (2), have been isolated from the roots of Saussurea lappa C. Their structures, including the absolute configurations, were elucidated by spectroscopic methods.

The Xenopus arx gene is expressed in the developing rostral forebrain.

The human aristaless-related homeobox ( ARX) gene is mutated in several patients with X-linked mental retardation and/or other neurologic pathologies. We report the isolation and expression pattern of a Xenopus arx gene. Similar to other vertebrate arx genes, Xenopus arx is expressed in the developing telencephalon, diencephalon, and floor plate.

Overexpression of insulin receptor substrate-1, but not insulin receptor substrate-2, protects a T cell hybridoma from activation-induced cell death.

The insulin receptor substrate (IRS) family of signaling molecules is expressed in lymphocytes, although their functions in these cells is largely unknown. To investigate the role of IRS in the protection of T cells from activation-induced cell death (AICD), we transfected the T cell hybridoma A1.1, which is IL-4 responsive but lacks expression of IRS family members with cDNA encoding IRS1 or IRS2. Stimulation of these clones with immobilized anti-CD3-induced expression of CD69 to the same level as the parental A1.1 cells. However, the A1.1 IRS1-expressing cells were markedly resistant to AICD, while the A1.1 IRS2-expressing cells were not. Inhibition of phosphatidylinositol 3'-kinase in the A1.1 IRS1-expressing cells did not abrogate their resistance to AICD. Fas mRNA was induced similarly by anti-CD3 in A1.1, A1.1 IRS1-expressing, and A1.1 IRS2-expressing cells. However, induction of Fas ligand (FasL) mRNA and functional FasL protein was delayed and decreased in IRS1-expressing cells, but not in IRS2-expressing cells. The induction of transcription from a 500-bp FasL promoter and a minimal 16-mer early growth response element linked to luciferase was also impaired in the IRS1-expressing cells. These results suggest that overexpression of IRS1, but not IRS2, protects A1.1 cells from AICD by diminishing FasL transcription through a pathway that is independent of the tyrosine phosphorylation of IRS1 and phosphatidylinositol 3'-kinase activity.