Abstract Introduction Non-small cell lung cancer (NSCLC) is a malignancy of the epithelial cells in the lung originating from the central bronchi spreading to the terminal alveoli. It is the most common form, accounting for 85% of diagnoses. The low 5-year survival rate of 28% is due to acquired treatment resistance to standard of care anti PD-L1 immunotherapy. Therefore, it is pertinent to understand NSCLC immune signaling and fully describe cellular niches within the tumor microenvironment. Sensory neurons of the peripheral nervous system (PNS) have been shown to modulate immune responses across cancer types. Increased innervation and neuropeptide secretion is associated with increased tumor burden and dysregulated immune cell function. Neuropeptide substance P (SP) has been shown to drive breast cancer metastasis, and sensory neurons contribute to regulating immune responses in allergies, homeostasis, and infection. Methods In a tumor injectable mouse model of NSCLC with KRAS and p53 mutations (KP), lung tissue was broad cell immunophenotyped, and immunofluorescently stained with neuronal, immune, and tumoral markers. Cell supernatant was used for neuropeptide ELISA and cytokine analysis. This was performed in C57BL/6 and Tac1KO mice. Results SP immunofluorescence staining and ELISA showed increased expression in KP injected mice compared to control. When the SP-encoding gene Tac1 is knocked out, tumor burden is increased. We hypothesize this is due to dysregulated immune cell populations, particularly macrophages. Conclusion To approach NSCLC from a new perspective, we probe the impact of sensory neurons and neuropeptides on immune response and tumor burden in a KP tumor injectable mouse model, where we knockout neuropeptide SP. To better understand potential mechanism and neuron induced modulation of the tumor microenvironment, we plan to investigate neuropeptide level expression and immunophenotyping within the tumor injectable mouse model, human patient samples, and human co-culture in vitro models of NSCLC. Funding Source R01HL165259-01A, R01HL165259-03S2 Topic Categories Neuroimmunology (NEUR)

Abstract Introduction Chronic poor wound healing is a significant global health burden, contributing to morbidity and mortality. Natural killer (NK) cells are innate lymphoid cells that possess both cytotoxic and infection-clearing roles. In the context of tissue repair, NK cells have been shown to have interactions with endothelial cells in the modulation of fibrosis and in the process of re-epithelialization. Consequently, NK cells may have the potential to influence the process of wound healing. The few studies that have examined NK cells’ role in wound healing have generated conflicting results, with one study reporting that NK cell ablation accelerates wound closure while another stated that NK cells have a positive role by limiting the acute inflammatory reaction to wounding. We investigate the role of NK cells as regulators of wound healing using multiple mouse models of skin injury. Methods Our data established the kinetics of NK cell recruitment in the wound with flow cytometry and characterized a unique phenotype that wound NK cells possess. Two mouse models of wound closure, a dorsal punch biopsy and tail-excisional wound, were used in conjunction with a NK-depleting antibody to measure the rate of wound closure in the absence of NK cells. Results The tail-excisional model, which heals primarily by re- epithelialization, demonstrated that wound closure is slower in the absence of NK cells. However, in the punch biopsy model, which relies on the contraction of skin to heal, NK depletion had no significant impact on the rate of wound closure. Using a model that allowed for the analysis of wound cells and fluid, we found that the growth factors EGF and VEGF were increased in the absence of NK cells. RNAseq data of NK-depleted mice showed the overexpression of collagen production genes related to skin and vasculature development. Conclusion This work contributes to the wound healing field by furthering insights regarding NK cells’ influence on wound closure, re-epithelialization, and vascularization. Funding Source Defense Advanced Research Projects Agency (DARPA), Dean’s Areas of Emerging New Science Award (Brown U.), NHLBI R01HL126887, Carney Institute Innovation Awards, R01HL165259-01A1 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Abstract Introduction Down Syndrome (DS) is the most prevalent chromosomal abnormality worldwide, affecting 1 in every 700 live births. Although typically noted for its neurological implications, individuals with DS exhibit severe pulmonary malformations as well, including poor branching, impaired alveolar formation, and low lung mass. The triplication of chromosome 21 (HSA21) in DS results in the overexpression of several immunological genes found on HSA21, including four of the six interferon (IFN) receptor genes. Increased IFN receptor expression constitutively activates the IFN signaling pathway, fostering an antiproliferative environment during development. Given that dysregulated IFN signaling has been linked to impaired cardiogenesis in DS mouse models, we hypothesize that hyperactive IFN signaling similarly impairs human pulmonary development. Methods Although mechanistically useful, animal models prove incomplete in fully recapitulating a chromosomal disorder such as DS. Thus, we have utilized Trisomy HSA21 (T21) induced pluripotent stem cells (iPSCs) to establish a DS Branching Lung Organoid (BLO) model that recapitulates key hallmarks of pulmonary development, including branching, budding, and epithelial differentiation. Using this model, we aim to investigate how dysregulated IFN signaling contributes to pulmonary malformation in DS. Results iPSCs derived from an individual mosaic for DS, providing both diploid (D21) and triploid (T21) isogenic cell lines, were used to generate BLOs. We hypothesize that T21 induces impairments to healthy BLO morphogenesis that can be corrected when proper IFN signaling is restored. Conclusion Given that the lungs continue to form through early childhood, interventions given to infants immediately following birth have displayed efficacy in facilitating healthy pulmonary development. Thus, studying how lung malformations arise in the development of DS may offer insight into restorative therapies for these individuals, ultimately curbing high pulmonary associated mortality. Funding Source R01HL165259-03S1 Topic Categories Cytokines and Chemokines and their Receptors (CCR)

Abstract Introduction Lung cancer remains a leading cause of cancer mortality, with non-small cell lung carcinoma (NSCLC) comprising over 80% of cases. While immunotherapies have improved survival, challenges persist in overcoming tumor-driven immunosuppression and sustaining effective immune activation. Emerging evidence suggests the lung microbiome may influence these immune processes, yet its role in shaping NSCLC progression remains poorly defined. Methods Three human lung microbiome datasets were analyzed to identify microbial features of NSCLC, controlling for tissue type, therapy, smoking, and antibiotic use. Reads were processed with DADA2 and annotated via KEGG and MetaCyc. Two murine models were used: a K-Ras mutant model induced by intratracheal Cre recombinase, and xenografts (KP-NINJA, KP-HELLO) expressing neoantigens to recruit immune subsets. Mice were exposed intratracheally to heat-inactivated Burkholderia (health-associated), Veillonella dispar (disease-associated), or Klebsiella LPS (control). Results Human NSCLC tissues showed significantly reduced microbial diversity and metabolic activity, indicating a loss of microbial integrity with disease. In mice, immunotherapy preserved microbial diversity and structure in proportion to tumor regression, while neoantigen-expressing tumors that promoted immune infiltration similarly restored microbial balance. Direct administration of health-associated microbes enhanced immune cell infiltration and reduced tumor burden, whereas disease-associated microbes suppressed immune engagement and promoted tumor persistence. Spatial mapping revealed microbial byproducts colocalized with innate immune cells, supporting their role in amplifying anti-tumor immunity through local signaling networks. Conclusion These findings uncover a dynamic interplay between lung microbes and tumor immunity. Modulating microbial composition can reshape immune responses, presenting a novel strategy to improve NSCLC immunotherapy outcomes. Funding Source NIH T32 Brown Respiratory Research Training Program, NHLBI R01HL126887, NIH/NIEHS 5T32ES007272-24 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Introduction Lung infections represent a major global health burden, with pneumonia remaining one of the leading causes of death in the United States. Secondary bacterial pneumonia following infection with influenza A virus (IAV) significantly increases disease severity and mortality, largely due to disruption of host resistance and tolerance mechanisms. Among bacterial pathogens, Streptococcus pneumoniae (S.p) is most commonly associated with influenza co-infection, a condition characterized by excessive inflammation, tissue damage, and poor therapeutic outcomes. Macrophages, as key orchestrators of pulmonary immune responses, regulate both pathogen clearance and inflammation through tightly controlled cell death pathways. Indeed, the progression of infection relies on the ability of host cells to trigger the appropriate programmed cell death response, which is usually inflammatory (pyroptosis and necroptosis). Pathogens redirect host cell death signaling towards cell death modalities that favor their adaptation, usually anti-inflammatory (apoptosis). These mechanisms lead to the occurrence of PANoptosis, a programmed cell death that incorporates elements of pyroptosis, apoptosis, and necroptosis and where caspase-8 serves as a central regulator. Methods To study the role of caspase-8 in co-infection by IAV and S.p, we infected macrophages derived from mouse bone marrow and human macrophages (THP1 cells) with a specific deletion of caspase-8 obtained using the lox cre and CRISPR methods, respectively. Results We found that the loss of caspase-8 led to a reduction in bacterial growth but an increase in necroptotic cell death, in a manner dependent on RIPK3 and MLKL. Furthermore, caspase-8-deficient macrophages showed reduced production of IL-1β and pro-inflammatory cytokines. Conclusion These results indicate that caspase-8 plays a protective and regulatory role during co-infection by IAV and S.p. by coordinating macrophage PANoptosis through the control of inflammation and pathogen replication. Funding Source n/a Topic Categories Viral Immunology (VIR)

Abstract Introduction Individuals recovering from surgery are at risk for nosocomial pneumonia. When this occurs, the innate immune system is faced with competing inflammatory sites in the injured skin and the infected lung, raising the question of how the immune response prioritizes its response. We hypothesized that a lung infection would disrupt the normal course of wound healing. Methods The impact of nosocomial pneumonia on laparotomy healing was assessed using data obtained from the ACS National Surgical Quality Improvement Program database. Mouse models were also developed to assess the impact of post-operative pulmonary infection on the innate immune response. Tail skin excision was followed by pulmonary K. oxytoca infection to measure the effect on rate of wound closure. Wounding by the dorsal subcutaneous implantation of polyvinyl alcohol sponges was followed by pulmonary K. oxytoca infection to assess cellular responses at the wound site. Results Laparotomy data showed that patients with pneumonia had higher rates of wound dehiscence. In mice, lung infection slowed skin wound closure and impaired wound innate immune responses. Wound fluid IL-1b and downstream chemokines were rapidly suppressed following lung infection. Local administration of rIL-1b and chemokines in mice with lung infection rescued wound inflammation, but at the expense of lung bacterial clearance. IL-1 is regulated by signaling inhibitors. IL-1RA, a non-signaling ligand of the IL-1 receptor, was upregulated in the bronchoalveolar lavage fluid and plasma of lung-infected mice. Lung infection also increased the expression of the IL-1 receptor decoy, IL-1R2, on blood and wound neutrophils. Administration of IL-1RA to wounded mice phenocopied the effects of lung infection. Conclusion These results show that the infected lung induces systemic IL-1 signaling inhibitors as a protective mechanism to tune distal inflammation, preventing leukocyte migration to the wound and allowing the innate immune system to prioritize inflammatory sites. Funding Source Defense Advanced Research Projects Agency (DARPA), Dean’s Areas of Emerging New Science Award (Brown U.), NIEHS T32-ES7272 (Training in Environmental Pathology), NIGMS COBRE Award P20GM10935, R01HL126887, R01HL165259-01A1, Carney Institute Innovation Awards Topic Categories Cytokines and Chemokines and their Receptors (CCR)

This study examines immune and inflammatory responses in draining wound fluid over the course of the early stages of wound healing in patients recovering from spinal fusion surgery. The inflammatory phase of wound healing is essential for setting the stage for successful tissue repair and preventing chronic or poorly healing wounds. Scoliosis can be idiopathic or occur secondary to neuromuscular disorders, which are known to be associated with poor wound healing outcomes. We hypothesised that neuromuscular scoliosis patients would exhibit differences in inflammatory wound healing markers compared to idiopathic scoliosis patients. Comparison of the cellular and cytokine contents of draining wound fluid revealed that several inflammatory cytokines were elevated in the neuromuscular scoliosis patient group compared to idiopathic, whereas the leukocyte contents were the same between groups. This study shows that draining wound fluid is a good source of cellular and soluble biomarkers for acute wound healing and can be used to determine changes in individuals at risk for wound healing complications.

Wound healing necessitates a balance between synthesis and breakdown of extracellular matrix components, which is tightly regulated by proteases and their inhibitors. While studies have demonstrated that citric and acetic acid treatments enhance healing in recalcitrant wounds, the underlying proteolytic mechanisms remain elusive. In this study, we systematically evaluated changes in the proteolytic activity of murine wound fluid upon acidification. A library of 228 synthetic peptides served as reporters of protease activity at pH 7.4, pH 5.0, and pH 3.5. The peptide digestion patterns differed at each pH, revealing that proteases active at pH 7.4 are inactivated at pH 3.5. Notably, cathepsin D emerged as the dominant active enzyme at pH 3.5, and its activity was inhibited by pepstatin. Using a fluorogenic substrate, we quantified cathepsin D activity across varying pH levels and demonstrated optimal activity between pH 3.0 and 3.8. This activity was detectable as early as 1 day postinjury and persisted over the following 10 days. Importantly, human wound fluid exhibited the same activity profile, validating the mouse model as a relevant system for studying acid-mediated wound healing processes.

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated Ca V 2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin. Here we report that the cytokine interleukin-1α (IL-1α), an alarmin, is necessary and sufficient to trigger rapid heat and mechanical hypersensitivity in skin. Of 20 cytokines screened, only IL-1α was consistently detected in hind paw interstitial fluid in response to intradermal capsaicin and, similar to behavioral sensitivity to heat, IL-1α levels were also dependent on peripheral Ca V 2.2 channel activity. Neutralizing IL-1α in skin significantly reduced capsaicin-induced changes in hind paw sensitivity to radiant heat and mechanical stimulation. Intradermal IL-1α enhances behavioral responses to stimuli and, in culture, IL-1α enhances the responsiveness of Trpv1 -expressing sensory neurons. Together, our data suggest that IL-1α is the key cytokine that underlies rapid and reversible neuroinflammatory responses in skin.

Spectral fingerprinting has emerged as a powerful tool, adept at identifying chemical compounds and deciphering complex interactions within cells and engineered nanomaterials. Using near-infrared (NIR) fluorescence spectral fingerprinting coupled with machine learning techniques, we uncover complex interactions between DNA-functionalized single-walled carbon nanotubes (DNA-SWCNTs) and live macrophage cells, enabling in situ phenotype discrimination. Through the use of Raman microscopy, we showcase statistically higher DNA-SWCNT uptake and a significantly lower defect ratio in M1 macrophages as compared to M2 and naïve phenotypes. NIR fluorescence data also indicate that distinctive intra-endosomal environments of these cell types give rise to significant differences in many optical features such as emission peak intensities, center wavelengths, and peak intensity ratios. Such features serve as distinctive markers for identifying different macrophage phenotypes. We further use a support vector machine (SVM) model trained on SWCNT fluorescence data to identify M1 and M2 macrophages, achieving an impressive accuracy of > 95%. Finally, we observe that the stability of DNA-SWCNT complexes, influenced by DNA sequence length, is a crucial consideration for applications such as cell phenotyping or mapping intra-endosomal microenvironments using AI techniques. Our findings suggest that shorter DNA-sequences like GT 6 give rise to more improved model accuracy (> 87%) due to increased active interactions of SWCNTs with biomolecules in the endosomal microenvironment. Implications of this research extend to the development of nanomaterial-based platforms for cellular identification, holding promise for potential applications in real time monitoring of in vivo cellular differentiation. TOC Graphic:

Wound healing necessitates a balance between synthesis and breakdown of extracellular matrix components, which is tightly regulated by proteases and their inhibitors. Studies have shown that treatment of poorly healing wounds with acid results in improved healing. In this study, we systematically evaluated changes in proteolytic activity of murine wound fluid upon acidification. A library of 228 synthetic peptides served as reporters of protease activity at pH 7.4, pH 5.0 and pH 3.5. The peptide digestion patterns differed at each pH, revealing that proteases active at pH 7.4 are inactivated at pH 3.5. Notably, aspartic acid proteases emerged as the dominant active enzymes at pH 3.5 and their activity was inhibited by pepstatin. Using a fluorogenic substrate, we quantified aspartic protease activity across varying pH levels and demonstrated optimal activity between pH 3.0 and 3.8. This activity was detectable as early as one day post-injury and persisted over the following ten days. Importantly, human wound fluid exhibited the same activity profile, validating the mouse model as a relevant system for studying acid-mediated wound healing processes. ### Competing Interest Statement The authors have declared no competing interest.

Neuroinflammation can lead to chronic maladaptive pain affecting millions of people worldwide. Neurotransmitters, cytokines, and ion channels are implicated in neuroimmune cell signaling, but their roles in specific behavioral responses are not fully elucidated. Voltage-gated CaV2.2 channel activity in skin controls rapid and transient heat hypersensitivity induced by intradermal (i.d.) capsaicin via IL-1 alpha cytokine signaling. CaV2.2 channels are not, however, involved in mechanical hypersensitivity that developed in the i.d. capsaicin animal model. Here, we show that CaV2.2 channels are also critical for heat hypersensitivity induced by i.d. complete Freund adjuvant (CFA). i.d. CFA, a model of chronic neuroinflammation, involves ongoing cytokine signaling for days leading to pronounced edema and hypersensitivity to sensory stimuli. Peripheral CaV2.2 channel activity in the skin was required for the full development and week-long time course of heat hypersensitivity induced by i.d. CFA, but paw edema and mechanical hypersensitivity were independent of CaV2.2 channel activity. CFA induced increases in several cytokines in hindpaw fluid including IL-6 which was also dependent on CaV2.2 channel activity. Using IL-6-specific neutralizing antibodies in vivo, we show that IL-6 contributes to heat hypersensitivity and that neutralizing both IL-1 alpha and IL-6 was even more effective at reducing the magnitude and duration of CFA-induced heat hypersensitivity. Our findings demonstrate a functional link between CaV2.2 channel activity and the release of IL-6 in the skin and show that CaV2.2 channels have a privileged role in the induction and maintenance of heat hypersensitivity during chronic forms of neuroinflammation in the skin.

Spectral fingerprinting has emerged as a powerful tool that is adept at identifying chemical compounds and deciphering complex interactions within cells and engineered nanomaterials. Using near-infrared (NIR) fluorescence spectral fingerprinting coupled with machine learning techniques, we uncover complex interactions between DNA-functionalized single-walled carbon nanotubes (DNA-SWCNTs) and live macrophage cells, enabling in situ phenotype discrimination. Utilizing Raman microscopy, we showcase statistically higher DNA-SWCNT uptake and a significantly lower defect ratio in M1 macrophages compared to M2 and naive phenotypes. NIR fluorescence data also indicate that distinctive intraendosomal environments of these cell types give rise to significant differences in many optical features, such as emission peak intensities, center wavelengths, and peak intensity ratios. Such features serve as distinctive markers for identifying different macrophage phenotypes. We further use a support vector machine (SVM) model trained on SWCNT fluorescence data to identify M1 and M2 macrophages, achieving an impressive accuracy of >95%. Finally, we observe that the stability of DNA-SWCNT complexes, influenced by DNA sequence length, is a crucial consideration for applications, such as cell phenotyping or mapping intraendosomal microenvironments using AI techniques. Our findings suggest that shorter DNA-sequences like GT(6) give rise to more improved model accuracy (>87%) due to increased active interactions of SWCNTs with biomolecules in the endosomal microenvironment. Implications of this research extend to the development of nanomaterial-based platforms for cellular identification, holding promise for potential applications in real time monitoring of in vivo cellular differentiation.

Infection by the Influenza A Virus (IAV) remains a persistent issue contributing to increased risk of morbidity and mortality, especially for susceptible individuals. IAV infection can lead to increased susceptibility to bacterial infections, which further exacerbate disease. Therapeutics targeting pathogens are often ineffective as pathogens can mutate to avoid potential vaccines and antimicrobials. Improving our understanding of IAV/bacteria-host interactions can allow us to improve host-specific approaches to tackle potential morbidity and mortality brought on by coinfections that are primarily found in epithelial cells. Lung epithelial cells not only serve as an integral part of the respiratory system, but are also crucial in host defense against respiratory infections. Therefore, we adopted an Air-Liquid Interface (ALI) strategy for the culture of human bronchial epithelial cells (HBEC) to mimic the structure and function of respiratory epithelial tissue. ALI cultures were then subjected to single and coinfections by IAV and Streptococcus pneumoniaeto assess the resulting pathology. We discovered that the specific type of infection influenced the type of programmed cell death that occurred, ranging from apoptotic to necroptotic. Additionally, coinfection with IAV and bacteria caused a unique gene expression profile. These findings demonstrate that the presence of specific pathogens causes a significant change in the cell death characteristics as well as transcriptional profile of lung epithelial cells. We hope that the progress of our investigation further elucidates consequences of host-pathogen interactions that can contribute to developing novel therapeutic strategies for complex lung infections. Supported by grants from the NIH, NHLBI R01HL126887 (A.J.), P20GM121344 Pilot Project (A.J.), and under award number RL5GM118975 (R.Z.) of NIGMS.

Individuals recovering from surgery are at risk of nosocomial infections such as pneumonia. In this setting, the innate immune system faces competing inflammation in the injured skin and infected lung, raising the question of how the immune response prioritizes inflammatory sites. We hypothesized that the initiation of a lung infection would disrupt wound healing. Supporting this hypothesis, data from the ACS NSQIP database showed that the rate of laparotomy dehiscence in surgical patients with pneumonia was over double the rate of those without pneumonia. We next developed mouse models of post-operative pulmonary infection to assess cellular mechanisms. Using the tail skin excision and dorsal subcutaneous PVA sponge implantation wound healing models, we found that bacterial lung infection slowed the rate of wound closure and impaired wound neutrophil, monocyte, and cytokine/chemokine responses. Lung infection quickly suppressed wound fluid IL-1b and downstream chemokine concentrations within 6 hours of inoculation. IL-1 receptor antagonist (IL-1RA), an inhibitor of IL-1 signaling, was upregulated in the bronchoalveolar lavage fluid and plasma 6 hours after lung infection. Administration of IL-1RA to the wounds of uninfected mice phenocopied the effects of lung infection. Conversely, rIL-1b rescued wound inflammation when administered to the to the wounds of mice with lung infection, although this occurred at the expense of lung bacterial clearance. These results suggest that the infected lung induces IL-1RA systemically as a protective mechanism to tune inflammation at distal sites via IL-1b suppression, allowing the innate immune system to prioritize inflammatory insults. Supported by grants from Defense Advanced Research Projects Agency (DARPA), Brown University (Dean's Areas of Emerging New Science Award, Carney Institute Innovation Awards), and NIH (NIES T32-ES7272 Training in Environmental Pathology; NIGMS COBRE Award P20GM10935, NHLBI R01 126887),

The lung is a complex and unique organ system whose biology is strongly influenced by environmental exposure, oxygen abundance, connection to extrapulmonary systems via a dense capillary network, and an array of immune cells that reside in the tissue at steady state. The lung also harbors a low biomass community of commensal microorganisms that are dynamic during both health and disease with the capacity to modulate regulatory immune responses during diseases such as cancer. Lung cancer is the third most common cancer worldwide with the highest mortality rate amongst cancers due to the difficulty of an early diagnosis. This review discusses the current body of work addressing the interactions between the lung microbiota and the immune system, and how these two components of the pulmonary system are linked to lung cancer development and outcomes. Bringing in lessons from broader studies examining the effects of the gut microbiota on cancer outcomes, we highlight many challenges and gaps in this nascent field.

Individuals who are hospitalized with traumatic injuries are susceptible to pneumonia. The effect of lung infection on the course of wound healing is not well understood in this setting. To begin to understand this, we examined data from patients who had received laparotomies and found that those who developed pneumonia experienced higher rates wound dehiscence compared to those without pneumonia. Based on this finding and the known role for the innate immune system in responding to injuries and infections, we hypothesized that the immune system cannot meet the demands of competing inflammatory insults in the skin and lungs, leading to impairment of one or both responses. To test this, we established two murine models of post-injury lung infection in which mice are wounded by the dorsal subcutaneous implantation of polyvinyl alcohol (PVA) sponges or by tail skin excision, then infected intranasally with Klebsiella oxytoca. In these models, the presence of a wound did not alter the response to pulmonary infection. In contrast, pulmonary infection delayed the closure tail skin wounds. Pulmonary infection also led to rapid suppression of IL-1beta and chemokine levels, as well as decreased leukocyte accumulation, in PVA sponge wounds. The impaired wound healing observed in infected mice could be rescued by the addition of IL-1beta or the chemokines CCL2 and CXCL1 to the wound bed; however, these treatments caused a delay in bacterial clearance in the lungs. These data suggest that the immune response is not fully equipped to respond to disparate and competing inflammatory insults, which could have implications beyond the setting of post-injury pneumonia. Supported by grants from NIH (NIES T32-ES7272, NIGMS COBRE Award P20GM10935, and NHLBI R01HL126887), Defense Advanced Research Projects Agency (DARPA), and Brown University (Dean’s Areas of Emerging New Science Award and Carney Institute Innovation Awards)

Annually in the U.S, the cost to treat the more than 6 million sufferers of chronic, non-healing wounds exceeds 50 billion U.S.D. Despite extensive characterization of the functionally diverse cellular and molecular constituents found in the wound bed, wound healing therapies are lacking. The pro- and anti-inflammatory capabilities of the innate immune system make it critical in the wound healing process. Natural killer (NK) cells are innate lymphocytes which exhibit similar pro- and anti-inflammatory capabilities. Despite the breadth of their functions, ranging from anti-microbial to maintaining a healthy pregnancy, there is a paucity of information regarding NK cells in the wound healing process. Using mouse wound models, we have identified 2 distinct populations of NK cells abundantly present in seven-day old sterile, cutaneous wounds. In addition, we have identified NK cells in the wound effluent of patients who underwent corrective surgery for idiopathic scoliosis. Previous work in different models suggests that these 2 NK cell populations exhibit proliferative effector functions related to their potential to facilitate organogenesis and T cell activation. Interestingly, these are both processes implicated in late stage wound healing. Using our mouse model of cutaneous wound healing we have shown that NK cell depletion delays closure. Furthermore, we show that NK cell depleted wounds demonstrate an aberrant growth factor response. With this work we aim to determine the impacts wound natural killer cell phenotype and function have on cutaneous wound healing outcomes and to determine the role of natural killer cells in wound growth factor expression.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global pandemic. In addition to the acute pulmonary symptoms of coronavirus disease (COVID-19) (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients. Currently, the molecular pathogenesis underlying COVID-19–associated coagulopathies are unknown. Identifying the molecular basis of how SARS-CoV-2 drives coagulation is essential to mitigating short- and long-term thrombotic risks of sick and recovered patients with COVID-19. We aimed to perform coagulation-focused transcriptome analysis of in vitro infected primary respiratory epithelial cells, patient-derived bronchial alveolar lavage cells, and circulating immune cells during SARS-CoV-2 infection. Our objective was to identify transcription-mediated signaling networks driving coagulopathies associated with COVID-19. We analyzed recently published experimentally and clinically derived bulk or single-cell RNA sequencing datasets of SARS-CoV-2 infection to identify changes in transcriptional regulation of blood coagulation. We also confirmed that the transcriptional expression of a key coagulation regulator was recapitulated at the protein level. We specifically focused our analysis on lung tissue–expressed genes regulating the extrinsic coagulation cascade and the plasminogen activation system. Analyzing transcriptomic data of in vitro infected normal human bronchial epithelial cells and patient-derived bronchial alveolar lavage samples revealed that SARS-CoV-2 infection induces the extrinsic blood coagulation cascade and suppresses the plasminogen activation system. We also performed in vitro SARS-CoV-2 infection experiments on primary human lung epithelial cells to confirm that transcriptional upregulation of tissue factor, the extrinsic coagulation cascade master regulator, manifested at the protein level. Furthermore, infection of normal human bronchial epithelial cells with influenza A virus did not drive key regulators of blood coagulation in a similar manner as SARS-CoV-2. In addition, peripheral blood mononuclear cells did not differentially express genes regulating the extrinsic coagulation cascade or plasminogen activation system during SARS-CoV-2 infection, suggesting that they are not directly inducing coagulopathy through these pathways. The hyperactivation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system in SARS-CoV-2–infected epithelial cells may drive diverse coagulopathies in the lung and distal organ systems. Understanding how hosts drive such transcriptional changes with SARS-CoV-2 infection may enable the design of host-directed therapeutic strategies to treat COVID-19 and other coronaviruses inducing hypercoagulation.

Since the emergence of the novel respiratory coronavirus, designated SARS-CoV-2 in 2019, there have been more than 2.5 million deaths worldwide, and the numbers continue to rise (Zhu et al., 2020). Symptoms from infection by SARS-CoV-2, range from entirely asymptomatic to severe acute respiratory distress syndrome (ARDS) (Wiersinga et al., 2020) (Yuki et al., 2020). Severe cases of COVID-19, the disease caused by SARS-CoV-2, are complicated by ARDS and hyper-inflammation, with many patients requiring mechanical ventilation and ICU admission due to hypoxia (Wiersinga et al., 2020) (Edler et al., 2020). Much of what we know about the disease comes from data from hospitalized patients, who have most likely been infected for a long time and often have severe symptoms. Understanding how SARS-CoV-2 infection influences the immune response early in the course of infection is of upmost importance to fully understand how the infection progresses. The manuscript by Liou et al. (2020) uses an innovative approach to examine the impact of early SARS-CoV-2 infection on the innate immune response in the upper respiratory system. By using nasal pharyngeal swab biospecimens from both SARS-CoV-2 positive patients and SARS-CoV-2 negative patients, the authors are able to determine the impact of a probable early infection on several factors that are important in the early response. In addition, by using both transcriptional and proteomic analysis, this study demonstrates how SARS-CoV-2 infection may influence both transcriptional and post-transcriptional regulation of inflammatory mediators. One of the most striking findings of the study is that there is a clear correlation of IP-10 (CXCL10) expression with viral load not only at the transcript level but also at the protein level. CXCL10 expression in the upper respiratory tract has been shown in previous studies to be a hallmark of a respiratory infection (Landry & Foxman, 2018). This chemokine attracts several different immune cell types to the site of infection including macrophages, Natural Killer cells, and activated T cells (Liu et al., 2011). CXCL10 is well known as a factor in ARDS progression, and it has been shown to be elevated in the lungs of patients infected with highly pathogenic avian influenza (HPAI) and SARS-CoV-1 (Jiang et al., 2005) (Peiris et al., 2004). Interestingly, it not only has chemotactic activity, but also controls apoptosis, cell growth and proliferation, and angiogenesis (Liu et al., 2011). CXCL10 has been shown to contribute to oxidative burst as well as chemotaxis of activated neutrophils in models of lung injury (Ichikawa et al., 2013). In COVID-19 patients a biomarker of disease severity is elevated levels of CXCL10 in the plasma (Yang et al., 2020) (Chen et al., 2020). Several previous studies using different models have also shown that nasal epithelial cells express CXCL10 (Gamage et al., 2020) (Lieberman et al., 2020). Another finding is that in many cases the protein levels do not correspond to what is seen at the mRNA level for the various inflammatory mediators looked at. However, proteins for CXCL10 and several interferon stimulated genes (ISGs) did correlate with transcript level. Further studies are necessary to determine mechanisms behind this interesting observation. It is unclear is this is due to a lack of translation or an active degradation process. Also, it is unknown if this a specific feature of SARS-CoV-2 infection where the virus is actively involved in the regulation of host inflammatory mediators, or if this is part of the host process in the tight regulation of the innate immune response during early infection. This study gives important insight into the early immune and anti-viral responses to initial SARS-CoV-2 infection of the upper respiratory tract. Understanding these early stages of infection will allow us to more fully understand the pathology of COVID-19.