Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Background: TLR4 polymorphism replacing Asp-299 with Gly and Thr-399 with Ile (D299G/T399I) causes LPS hyporesponsiveness. Results: TLR4 SNPs ⅐MD-2⅐LPS exhibits an agonistic 2:2:2 architecture. Local structural differences were observed around D299G, but not around T399I, SNP site. Conclusion: These local differences cause the modulation of surface properties of TLR4, which may affect ligand binding. Significance: This study provides structural evidence of the functionality of the mutant TLR4 carrying the SNPs.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as HI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV B-cell lymphoma.

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Toll-like receptor (TLR)4/MD-2, a sensor for LPS, delivers the MyD88-dependent signal from the cell surface, then traffics to endolysosomes and delivers the TRIF/TICAM-1-dependent signal. Both signals are thought to be dependent on cell surface TLR4/MD-2. Although TLR4/MD-2 is located also in recycling endosomes, the Golgi apparatus or the endoplasmic reticulum, little is known about a role for intracellular TLR4/MD-2 in LPS responses. We here studied intracellular LPS sensing in macrophages. PRAT4A (protein associated with TLR4 A) is a cochaperone for a general chaperone gp96 and required for cell surface expression of TLR4/MD-2. Cell surface TLR4/MD-2 was undetectable on PRAT4A(-/-) thioglycollate-elicited peritoneal macrophages (P-Macs) and bone marrow-derived macrophages (BM-Macs). LPS responses were all abolished in PRAT4A(-/-) P-Macs, whereas a part of LPS responses remained detectable in PRAT4A(-/-) BM-Macs. Of note, LPS responses in PRAT4A(-/-) BM-Macs were not necessarily dependent on TRIF/TICAM-1 signaling. PRAT4A(-/-) BM-Macs showed unimpaired production of both TRIF/TICAM-1-dependent chemokine RANTES (CCL5) and MyD88-dependent chemokine MCP-1 (CCL2). Moreover, up-regulation of co-stimulatory molecules, CD40 and CD86 was not altered. In contrast, TRIF/TICAM-1-dependent production of type I IFN was profoundly impaired. In response to heat-killed bacteria Escherichia coli, BM-Macs also required PRAT4A-independent TLR4/MD-2 for production of MCP-1 (CCL2) and RANTES (CCL5) and for up-regulation of CD40 and CD86, indicating that intracellular TLR4/MD-2 is able to sense phagocytosed bacteria and activate immune responses. These results demonstrate that intracellular TLR4/MD-2 is responsible for unique set of LPS responses.