The incidences of chronic inflammatory disorders have increased significantly over the past three decades1. Recent shifts in dietary consumption are believed to have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2cmo mice that express a homozygous L98P missense mutation in the Pombe Cdc15 homology (PCH) family proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) phosphatase spontaneously develop osteomyelitis that resembles chronic recurrent multifocal osteomyelitis (CRMO) in humans2-4. Recent reports demonstrated osteomyelitis to critically rely on IL-1β, but deletion of the inflammasome components caspase-1 and NLRP3 failed to rescue Pstpip2cmo mice from inflammatory bone disease5,6. Thus, the upstream mechanisms controlling IL-1β production in Pstpip2cmo mice remain to be identified. In addition, the environmental factors driving IL-1β-dependent inflammatory bone erosion are unknown. Here, we show that the intestinal microbiota of diseased Pstpip2cmo mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2cmo mice that were fed a diet rich in fat and cholesterol maintained a normal body weight, but were markedly protected against inflammatory bone disease and bone erosion. Diet-induced protection against osteomyelitis was accompanied by marked reductions in intestinal Prevotella levels and significantly reduced proIL-1β expression in distant neutrophils. Furthermore, proIL-1β expression was also decreased in antibiotics-treated Pstpip2cmo mice, and in wildtype mice that were kept under germfree conditions. We further demonstrated that combined deletion of caspases 1 and 8 was required for protection against IL-1β-dependent inflammatory bone disease, whereas deletion of each caspase alone, elastase or neutrophil proteinase-3 failed to prevent inflammatory disease. Collectively, this work reveals diet-associated changes in the intestinal microbiome as a critical factor regulating inflammasome- and caspase-8-mediated maturation of IL-1β and osteomyelitis in Pstpip2cmo mice.

IntroductionReceptor tyrosine kinases (RTKs) bind extracellular growth factors and activate intracellular signaling networks. The magnitude and kinetics of RTKs activation are tightly regulated, since they determine the quality and extent of the biologic response. 1 Attenuation of RTK signaling occurs by endocytosis and subsequent protein degradation. [2][3][4] Cbl proteins have been shown to be central players in these processes. [5][6][7] The members of the Cbl family, c-Cbl, Cbl-b, and Cbl-3, contain an N-terminal phosphotyrosine-binding (PTB) domain that allows direct interaction with activated RTKs, and a RING finger domain that classifies Cbl proteins as E3 ubiquitin ligases. 5,[8][9][10] Ubiquitylation of RTKs is important for their internalization, endocytic sorting, and targeting for degradation. 2 In addition to their ubiquitin E3 ligase activity, Cbl proteins also associate with the endocytic machinery via their C-terminus by recruiting proteins such as CIN85 and endophilins. 11,12 However, not only are Cbl proteins important for RTK signal termination, but they also mediate positive RTK signaling events to downstream effectors. Upon phosphorylation, Cbl molecules bind signaling molecules including SHP-2, Gab2, and PI3-kinase. 13 In animal models, but not in human cancers, oncogenic forms of Cbl have been described that are characterized by loss of the E3 ubiquitin ligase activity. 14,15 It has been reported that these oncogenic mutants of Cbl interact with activated RTKs and function in a dominant-negative fashion. 16,17 Aberrant signaling by the type III RTK Flt3 is an important event in the pathogenesis of acute myeloid leukemia (AML). Flt3 strongly influences hematopoietic progenitor cell homeostasis and is highly expressed in AML. [18][19][20][21] Also, about one third of AML cases harbor somatic, activating Flt3 mutations that cause myeloid transformation. [21][22][23][24] In contrast to activating mutations, little is known about the potential functions of Flt3 in AML cases lacking Flt3 mutations. Also, the mechanisms of Flt3 signal regulation and the role of Cbl proteins in these processes remain undetermined, although it has been shown that Flt3 activation is followed by Cbl phosphorylation. 25 Here, we analyzed the role of c-Cbl in the internalization, ubiquitylation, and biologic functions of wild-type Flt3 (Flt3-WT) and the most commonly described Flt3 mutations in AML, internal tandem duplication (Flt3-ITD). We found that the inhibition of Cbl function severely disturbed Flt3 signal transduction kinetics by blocking Flt3 internalization and ubiquitylation. As a consequence, interference with Cbl function induced ligand-independent, autoactive biologic effects of Flt3. Also, we describe a novel E3-ligase inactivating c-Cbl mutation isolated from the blasts of one AML patient. This mutant Cbl protein interfered with the function of endogenous c-Cbl and displayed in vitro transforming activity in myeloid cells that was dependent on the presence of Flt3. To our knowledge, this represents the ...

Zhou et al. demonstrate a requirement for the Let-7–Lin28b axis regulating a shift in development between fetal liver and bone marrow B lymphocyte progenitors in the generation of B1 versus B2 B cells. Specifically, the transcription factor Arid3a, induced by Lin28b and a target of Let-7 miRNA, is sufficient to recapitulate fetal B cell development from bone marrow progenitors.

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.