SUMMARY It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn’s disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn’s disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFα and IFNγ and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.

Inhibitors of the JAK family of non-receptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. Here we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naïve murine CD4+ T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation, and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and of the Th17 cytokines IL-17A, IL-17F and IL-22 were blocked when naïve Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A-production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented activation of STAT1, induction of T-bet and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells, as well as innate immune cell signaling.

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Objective. The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function.Methods. Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively.Results. Edema, inflammation, and osteoclastmediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1؉, CD3؉, and RANKL؉ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner.Conclusion. These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.

Next-generation sequencing (NGS) has revolutionized the approach of studying sequence variation, and has been well described in the clinical laboratory setting for the detection of constitutional alterations, as well as somatic tumor-associated variants. It is increasingly recognized that post-zygotic somatic alteration can be associated with congenital phenotypic abnormalities. Variation within the PI3K/AKT/mTOR pathway, including PIK3CA, has been described in somatic overgrowth syndromes and vascular malformations. Detection of PIK3CA somatic alteration is challenging because of low variant allele frequency (VAF) along with the need to assay involved tissue, thus necessitating a highly sensitive methodology. Here we describe the utility of target hybrid capture coupled with NGS for the identification of somatic variation in the PIK3CA-related overgrowth spectrum (PROS) among 14 patients submitted for clinical testing. Assay detection of low allelic fraction variation is coverage dependent with >90% sensitivity at 400× unique read depth for VAF of 10%, and approaching 100% at 1000×. Average read depth among the patient dataset across PIK3CA coding regions was 788.4. The diagnostic yield among this cohort was 71%, including the detection of two PIK3CA alterations novel in the setting of PROS. This report expands the mutational scope and phenotypic attributes of PROS disorders.