Biology subjects

Stark, J. M.

Publications and source records attributed to Stark, J. M..

5 recordsLinked to original sources

bioRxiv2021

The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

molecular biology↗

bioRxiv2021

Intestinal helminth infection transforms the CD4+ T cell composition of the skin

Intestinal helminth parasites can alter immune responses to vaccines, other infections, allergens and autoantigens, indicating effects on host immune responses in distal barrier tissues. We herein show that C57BL/6 mice infected with the strictly intestinal nematode Heligmosomoides polygyrus have impaired capacity to initiate skin immune responses and develop skin-resident memory cells to mycobacterial antigens, both during infection and months after deworming therapy. Surprisingly, and in contrast to a previously noted loss of T cells in peripheral lymph nodes, the skin of worm-infected mice harboured higher numbers of CD4+ T cells compared to skin of uninfected controls. H. polygyrus-specific TH2 cells accumulated during infection and remained after worm expulsion. Accumulation of TH2 cells in the skin was associated with increased expression of the skin-homing chemokine receptors CCR4 and CCR10 on CD4+ T cells in blood and mesenteric lymph nodes draining intestinal tissues, indicating gut-to-skin trafficking of cells. In conclusion, we show that infection by a strictly intestinal helminth has long-term effects on immune cell composition and local immune responses to unrelated antigens in the skin, revealing a novel mechanism for T cell colonization and worm-mediated immunosuppression in this organ.

immunology↗

bioRxiv2021

Laboratory mice with a wild microbiota generate strong allergic immune responses

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early life microbial exposures impede the development of subsequent allergic disease. However, unambiguous evidence that microbes reduce the development of allergic disorders is still lacking. Recently developed wildling mice contain a rich and diverse commensal and encounter a repertoire of microbes typical of the wild, with pathogenic potential. Here, we probed the hygiene hypothesis by comparing the development of allergic inflammation in wildlings to that of genetically identical mice lacking diverse microbial exposure. We find that wildlings develop stronger allergic inflammation in response to house dust mites with allergic T cell responses driven not only by cognate peptide antigens, but also by innate cytokines. In all, the results suggest that high microbial content and diversity potentiates, rather than restricts, allergic immune responses. One sentence summaryStrong allergic inflammation in the face of rich and diverse microbial exposures

immunology↗

bioRxiv2021

Sublingual allergen immunotherapy with recombinant dog allergens prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH2 and TH17 cell responses

Allergy to dogs affects around ten percent of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. Herein, we established a mouse model of dog allergy and tested the efficacy of a recombinant protein containing Can f 1, f 2, f 4 and f 6 as a sublingual immune therapy (SLIT). Repeated inhalation of dog extracts induced infiltration of the airways by TH2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. However, dog allergen extracts also induced robust TH17 cell responses, which was associated with a high neutrophilic infiltration of the airways and promoted airway hyperresponsiveness more potently than HDM allergens. scRNA-Seq analysis of T helper cells responding to dog allergens identified several unique clusters with TH17 cells being hallmarked by the expression of several receptors including IL-17RE. Analysis of T cell receptors also depicted a high frequency of clones that were shared between TH17, TH2 and suppressive Treg cells, indicative of the plasticity of T helper cells in this model. Importantly, prophylactic SLIT reduced airway hyperresponsiveness and type 2-mediated inflammation in this model supporting the use of recombinant allergens in immune therapy.

immunology↗

bioRxiv2020

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

biochemistry↗