Tran, H. L. | Search bioRxiv

bioRxiv2020

Pulmonary infection interrupts acute cutaneous wound healing through disruption of chemokine signals

Studies of the immune response typically focus on single-insult systems, with little known about how multi-insult encounters are managed. Pneumonia in patients recovering from surgery is a clinical situation that exemplifies the need for the patient to mount two distinct immune responses. Examining this, we have determined that poor wound healing is an unreported complication of pneumonia in laparotomy patients. Using mouse models, we found that lung infection suppressed the trafficking of innate leukocytes to wounded skin, while pulmonary resistance to the bacterial infection was maintained. The dual insults caused distinct systemic and local changes to the inflammatory response, the most striking being a rapid and sustained decrease in chemokine levels at the wound site of mice with pneumonia. Remarkably, replenishing wound chemokine levels completely rescued the wound-healing rate in mice with a pulmonary infection. These findings have broad implications for understanding the mechanisms guiding the innate immune system to prioritize inflammatory sites. One Sentence SummaryChemokine-mediated signaling drives the prioritization of innate immune responses to bacterial pulmonary infection over cutaneous wound healing. HighlightsO_LIHuman laparotomy patients with pneumonia have an increased rate of incision dehiscence, and this observation can be recapitulated in mouse models of bacterial lung infections and skin wounds. C_LIO_LILung infection causes rapid and sustained suppression of skin wound chemokine and inflammatory cytokine production as well as leukocyte recruitment. C_LIO_LIUnique systemic shifts in the immune compartment occur with two inflammatory insults, including the cytokine/chemokine signature and the mobilization, recruitment, and phenotype of innate leukocytes. C_LIO_LIRestoration of chemokine signaling in the wounds of mice that have a lung infection results in increased neutrophil trafficking to the wound site and rescues the rate of healing. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/084442v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@861000org.highwire.dtl.DTLVardef@1847251org.highwire.dtl.DTLVardef@6d6438org.highwire.dtl.DTLVardef@1ce99c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation

Chemical modification of antibodies is one of the most important bioconjugations utilized by biologists and biotechnology. To date, the field has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently we have developed oxaziridine chemistry for highly selective and efficient sulfimide modification of methionine called redox-activated chemical tagging (ReACT). Here, we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and generally good reaction efficiencies with the panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than ten-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, likely because of their reduced access to water. There was also a ten-fold variation in stability depending on the nature of the oxaziridine, which we determined was inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs and antibody drug conjugate potencies were comparable to those reported for cysteine-maleimide modifications of trastuzumab. We also found our antibody drug conjugates to be potent in a breast cancer mouse xenograft model. These studies provide a roadmap for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation.

bioengineering↗