No abstract

Immune cells express multiple Toll-like receptors (TLRs) that are concomitantly activated by a variety of pathogen products. Although there is presumably a need to coordinate the expression and function of TLRs in individual cells, little is known about the mechanisms governing this process. We show that a protein associated with TLR4 (PRAT4A) is required for multiple TLR responses. PRAT4A resides in the endoplasmic reticulum, and PRAT4A knockdown inhibited trafficking of TLR1 and TLR4 to the cell surface and ligand-induced trafficking of TLR9 to lysosomes. Other cell-surface molecules were expressed normally on immunocytes from PRAT4A−/− mice. There was impaired cytokine production to TLR ligands, except to the TLR3 ligand poly(I:C), and to whole bacteria. Activation of antigen-specific T helper type 1 responses were also defective. Moreover, PRAT4A−/− bone marrow chimeric mice were resistant to lipopolysaccharide-induced sepsis. These results suggest that PRAT4A regulates the subcellular distribution and response of multiple TLRs and is required for both innate and adaptive immune responses.

Toll-like receptors (TLRs) 3, 7, and 9 recognize microbial nucleic acids in endolysosomes and initiate innate and adaptive immune responses. TLR7/9 in dendritic cells (DCs) also respond to self-derived RNA/DNA, respectively, and drive autoantibody production. Remarkably, TLR7 and 9 appear to have mutually opposing, pathogenic or protective, impacts on lupus nephritis in MRL/lpr mice. Little is known, however, about the contrasting relationship between TLR7 and 9. We show that TLR7 and 9 are inversely linked by Unc93B1, a multiple membrane-spanning endoplasmic reticulum (ER) protein. Complementation cloning with a TLR7-unresponsive but TLR9-responsive cell line revealed that amino acid D34 in Unc93B1 repressed TLR7-mediated responses. D34A mutation rendered Unc93B1-deficient DCs hyperresponsive to TLR7 ligand but hyporesponsive to TLR9 ligand, with TLR3 responses unaltered. Unc93B1 associates with and delivers TLR7/9 from the ER to endolysosomes for ligand recognition. The D34A mutation up-regulates Unc93B1 association with endogenous TLR7 in DCs, whereas Unc93B1 association with TLR9 was down-regulated by the D34A mutation. Consistently, the D34A mutation up-regulated ligand-induced trafficking of TLR7 but down-regulated that of TLR9. Collectively, TLR response to nucleic acids in DCs is biased toward DNA-sensing by Unc93B1.

No abstract

Toll-like receptor 9 (TLR9) is an innate immune sensor for microbial DNA that erroneously responds to self DNA in autoimmune disease. To prevent autoimmune responses, Toll-like receptor 9 is excluded from the cell surface and silenced until the N-terminal half of the ectodomain (TLR9N) is cleaved off in the endolysosome. Truncated Toll-like receptor 9 (TLR9C) senses ingested microbial DNA, although the precise role of the truncation remains controversial. Here we show that TLR9 is expressed on the surface of splenic dendritic cells. Following the cleavage of TLR9 in the endolysosome, N-terminal half of the ectodomain remains associated with truncated TLR9, forming the complex TLR9N þ C. The TLR9-dependent cytokine production by Tlr9 À / À dendritic cells is rescued by a combination of TLR9N and TLR9C, but not by TLR9C alone. These results demonstrate that the TLR9N þ C complex is a bona fide DNA sensor.

Toll-like receptor 7 (TLR7) an innate immune sensor for microbial RNA, erroneously responds to self-derived RNA. To avoid autoimmune responses, TLR7 is suggested to be silenced until the N-terminal half of the TLR7 ectodomain (TLR7N) is cleaved off. Resultant truncated TLR7 (TLR7C) is thought to signal microbial RNA. We here show that TLR7N remains associated with TLR7C through a disulfide bond. By N-terminal amino acid sequencing, TLR7C was found to start at 461E or 462A. The newly established monoclonal anti-TLR7N showed that endogenous TLR7 in bone marrow-derived dendritic cells was almost all cleaved and cleaved TLR7N remained in endolysosomes. TLR7N in endolysosomes was linked with TLR7C by a disulfide bond. In contrast, TLR9 did not have a disulfide bond between TLR9N and TLR9C fragments. Among the cysteines unique to the ectodomain of TLR7 but not TLR9 (Cys98, Cys445, Cys475 and Cys722), Cys98 in TLR7N and Cys475 in TLR7C were required for an intramolecular disulfide bond. These cysteines were also needed for proteolytic cleavage of and RNA sensing by TLR7, but not for TLR7 trafficking from endoplasmic reticulum to endosomes. No response was seen in TLR7 mutants lacking the proteolytic cleavage site or TLR7C alone. These results demonstrate requirement for proteolytic cleavage and TLR7N in TLR7 responses and indicate RNA sensing by TLR7N + TLR7C.

Objectives:To describe the clinical features of children with ovotesticular disorder of sex development (DSD) and to review cases of ovotesticular DSD in Japan. Methods: Medical records of eight children diagnosed with ovotesticular DSD at our institute during the past 17 years were retrospectively evaluated. A review of 165 reported cases of ovotesticular DSD from Japanese institutions was carried out. Results: Mean follow up was 8.2 years for six children, with two children lost to follow up. Mean age at first presentation was 2.4 months. All children were Japanese. The most common initial manifestation was ambiguous genitalia. The female-: male ratio as the sex of rearing was 1:1. Gender reassignment, from male to female, was carried out in one child at 4-months-old. Genital surgery was always carried out in early childhood as per family desire. Appropriate gonadal tissue was preserved except for one child. No gonadal tumors were detected during follow up. Spontaneous pubertal development occurred in one boy. In reviewing Japanese data, the frequency of testes was higher than in other ethnicities and this was related to the higher incidence of 46,XY. Conclusions: According to our experience, most families in Japan desire early genital surgery in the case of ovotesticular DSD. Chromosomal and gonadal distributions in patients with ovotesticular DSD differ between Japanese and other ethnic groups. Treatment for these patients needs to be provided after considering the cultural and social backgrounds of DSD in Japan.

Prenatally detected hydronephrosis can worsen after spontaneous postnatal improvement. Long-term followup is not required after improvement of prenatal hydronephrosis. However, pediatric urologists need to tell the parents that the child should undergo ultrasonography if clinical symptoms develop.