Selection of silk-binding peptides by phage display

Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be useful for functionalization of silk for biomaterial applications

Role of the Lung Microenvironment in Regulating Adult and Neonatal AM Transcriptional Activity

Alveolar macrophages (AMs) play critical roles in metabolizing surfactant and protecting the lung against inhaled pathogens. AMs mature following the newborn period and their immaturity at birth may contribute to the variety of infectious and inflammatory lung diseases specifically affecting infants. However, the specific molecular features differentiating neonatal and adult AMs remain poorly understood. Here we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs that establish the molecular phenotypes specific to each developmental age. Adult AMs expressed higher levels of genes involved in lipid transport and metabolism (i.e. Fabp1, Pnpla5), consistent with their role in surfactant recycling. Neonatal AMs expressed higher levels of proinflammatory genes (Il1b, Il6, Tnf, and S100a8). ATAC-seq data detected adult AM peaks enriched with motifs recognizing KLF, GR, PPAR-gamma, and STAT. Accessible chromatin regions in neonatal AMs were highly enriched with binding motifs for the innate immunity transcription factors AP1, NF-kappa B, and IRF. To test how these baseline differences might impact AM innate immune function, we exposed neonatal and adult mice in vivo with inhaled LPS. AMs from both neonatal and adult mice demonstrated robust induction of Toll-Like Receptor 4 (TLR4) signaling pathways and similar patterns of chromatin accessibility. While neonatal and adult AMs did exhibit divergent expression patterns for some genes, the overall core innate response was similar. Intriguingly, neonatal AMs expressed higher basal levels of many LPS-induced genes, suggesting constitutive innate immune priming or activation in the neonatal lung. The lung microenvironment was a major factor regulating the unique molecular features of neonatal and adult AMs. Culturing isolated AMs for only 20h minimized the differences in gene expression between neonatal and adult cells. Interesting, while neonatal AMs in culture more closely resembled adult AMs, a core inflammatory signature of gene expression was retained. These data suggest that the proinflammatory phenotype of neonatal AMs results from both inherent properties of the cell and the lung microenvironment. Collectively, these studies provide new insights into the molecular mechanisms of lung innate immune development

Engineering Artificial Small RNAs for Conditional Gene Silencing in <i>Escherichia coli</i>

It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the <i>trans</i>-acting sRNAs in bacteria interact with the 5′ untranslated region (UTR) and/or the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and/or mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation. In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired endogenous genes in <i>Escherichia coli</i>. Using a fluorescent reporter gene that was translationally fused to a native 5′ mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a randomized antisense domain. Artificial sRNAs that posttranscriptionally repress two endogenous genes <i>ompF</i> and <i>fliC</i> were isolated and characterized. We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology

CRIg, a tissue-resident macrophage specific immune checkpoint molecule, promotes immunological tolerance in NOD mice, via a dual role in effector and regulatory T cells.

How tissue-resident macrophages (TRM) impact adaptive immune responses remains poorly understood. We report novel mechanisms by which TRMs regulate T cell activities at tissue sites. These mechanisms are mediated by the complement receptor of immunoglobulin family (CRIg). Using animal models for autoimmune type 1 diabetes (T1D), we found that CRIg+ TRMs formed a protective barrier surrounding pancreatic islets. Genetic ablation of CRIg exacerbated islet inflammation and local T cell activation. CRIg exhibited a dual function of attenuating early T cell activation and promoting the differentiation of Foxp3+ regulatory (Treg) cells. More importantly, CRIg stabilized the expression of Foxp3 in Treg cells, by enhancing their responsiveness to interleukin-2. The expression of CRIg in TRMs was postnatally regulated by gut microbial signals and metabolites. Thus, environmental cues instruct TRMs to express CRIg, which functions as an immune checkpoint molecule to regulate adaptive immunity and promote immune tolerance

Differential Immune Activation in Fetal Macrophage Populations.

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity

Developmental Immaturity of Siglec Receptor Expression on Neonatal Alveolar Macrophages Predisposes to Severe Group B Streptococcal Infection.

Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2-/- mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung