assessment-acute-wound-healing-using-dorsal-subcutaneous-polyvinyl

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Brown University

Secondary bacterial pneumonia leads to increased morbidity and mortality from influenza virus infections. What causes this increased susceptibility, however, is not well defined. Host defense from infection relies not only on immune resistance mechanisms but also on the ability to tolerate a given level of pathogen burden. Failure of either resistance or tolerance can contribute to disease severity, making it hard to distinguish their relative contribution. We employ a coinfection mouse model of influenza virus and Legionella pneumophila in which we can separate resistance and tolerance. We demonstrate that influenza virus can promote susceptibility to lethal bacterial coinfection, even when bacterial infection is controlled by the immune system. We propose that this failure of host defense is due to impaired ability to tolerate tissue damage.

Role of tissue protection in lethal respiratory viral-bacterial coinfection.

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

In order for vaccines to be effective within a given population not only do large numbers of people need to be vaccinated, but a large proportion of those vaccinated must develop protective immunity. The mechanisms that lead to a poor immune response to vaccination are complex and poorly understood, but include both genetic and environmental factors. The bacteria that exist throughout the human body, known as the microbiome, play a variety of roles in the development of the immune system. This is particularly true during infancy when the microbiome and the immune response are developing in tandem. Most vaccines are administered in early childhood to prevent outbreaks of devastating childhood diseases. Understanding the impact that the early microbiome plays in response to vaccination will improve our understanding of vaccine efficacy. 

Influence of the microbiome on response to vaccination.

Type I interferons (IFN-Is) are fundamental for antiviral immunity, but their role in bacterial infections is contradictory and incompletely described. Streptococcus pyogenes activates IFN-I production in innate immune cells, and IFN-I receptor 1 (Ifnar1)-deficient mice are highly susceptible to S. pyogenes infection. Here we report that IFN-I signaling protects the host against invasive S. pyogenes infection by restricting inflammation-driven damage in distant tissues. Lethality following infection in Ifnar1-deficient mice is caused by systemically exacerbated levels of the proinflammatory cytokine IL-1β. Critical cellular effectors of IFN-I in vivo are LysM+ and CD11c+ myeloid cells, which exhibit suppression of Il1b transcription upon Ifnar1 engagement. These cells are also the major source of IFN-β, which is significantly induced by S. pyogenes 23S rRNA in an Irf5-dependent manner. Our study establishes IL-1β and IFN-I levels as key homeostatic variables of protective, yet tuned, immune responses against severe invasive bacterial infection.

Type I Interferon Signaling Prevents IL-1β-Driven Lethal Systemic Hyperinflammation during Invasive Bacterial Infection of Soft Tissue.

Pathogen clearance and host resilience/tolerance to infection are both important factors in surviving an infection. Cells of the myeloid lineage play important roles in both of these processes. Neutrophils, monocytes, macrophages, and dendritic cells all have important roles in initiation of the immune response and clearance of bacterial pathogens. If these cells are not properly regulated they can result in excessive inflammation and immunopathology leading to decreased host resilience. Programmed cell death (PCD) is one possible mechanism that myeloid cells may use to prevent excessive inflammation. Myeloid cell subsets play roles in tissue repair, immune response resolution, and maintenance of homeostasis, so excessive PCD may also influence host resilience in this way. In addition, myeloid cell death is one mechanism used to control pathogen replication and dissemination. Many of these functions for PCD have been well defined in vitro, but the role in vivo is less well understood. We created a mouse that constitutively expresses the pro-survival B-cell lymphoma (bcl)-2 protein in myeloid cells (CD68(bcl2tg), thus decreasing PCD specifically in myeloid cells. Using this mouse model we explored the impact that decreased cell death of these cells has on infection with two different bacterial pathogens, Legionella pneumophila and Streptococcus pyogenes. Both of these pathogens target multiple cell death pathways in myeloid cells, and the expression of bcl2 resulted in decreased PCD after infection. We examined both pathogen clearance and host resilience and found that myeloid cell death was crucial for host resilience. Surprisingly, the decreased myeloid PCD had minimal impact on pathogen clearance. These data indicate that the most important role of PCD during infection with these bacteria is to minimize inflammation and increase host resilience, not to aid in the clearance or prevent the spread of the pathogen.

The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes.

Much research on infectious diseases focuses on clearing the pathogen through the use of antimicrobial drugs, the immune response, or a combination of both. Rapid clearance of pathogens allows for a quick return to a healthy state and increased survival. Pathogen-targeted approaches to combating infection have inherent limitations, including their pathogen-specific nature, the potential for antimicrobial resistance, and poor vaccine efficacy, among others. Another way to survive an infection is to tolerate the alterations to homeostasis that occur during a disease state through a process called host tolerance or resilience, which is independent from pathogen burden. Alterations in homeostasis during infection are numerous and include tissue damage, increased inflammation, metabolic changes, temperature changes, and changes in respiration. Given its importance and sensitivity, the lung is a good system for understanding host tolerance to infectious disease. Pneumonia is the leading cause of death for children under five worldwide. One reason for this is because when the pulmonary system is altered dramatically it greatly impacts the overall health and survival of a patient. Targeting host pathways involved in maintenance of pulmonary host tolerance during infection could provide an alternative therapeutic avenue that may be broadly applicable across a variety of pathologies. In this review, we will summarize recent findings on tolerance to host lung infection. We will focus on the involvement of innate immune responses in tolerance and how an initial viral lung infection may alter tolerance mechanisms in leukocytic, epithelial, and endothelial compartments to a subsequent bacterial infection. By understanding tolerance mechanisms in the lung we can better address treatment options for deadly pulmonary infections.

Surviving Deadly Lung Infections: Innate Host Tolerance Mechanisms in the Pulmonary System.

The innate immune system is responsible for many important functions in the body including responding to infection, clearing cancerous cells, healing wounds, and removing foreign substances. Although many of these functions happen simultaneously in life, most laboratory studies of the innate immune response focus on one activity. How the innate immune system responds to concurrent insults in different parts of the body is not well understood. This study explores the impact of a lung infection on the cutaneous wound healing process. We used two complimentary models of injury: the excisional tail wound and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. These models allow for assessment of the rate of closure and measurement of cellular and cytokine responses during acute wound healing, respectively. When mice with these healing wounds were infected with influenza A virus (IAV) in the lung there was a delay in wound healing. The viral lung infection suppressed the innate immune response in a healing wound, including cellular infiltrate, chemokines, growth factors, and cytokines. However, there was not a global immune suppression as there was an increase in inflammation systemically in mice with both infection and healing wounds compared to mice with only wounds or IAV infection. In addition, the lung immune response was largely unaffected indicating that responding to a lung infection is prioritized over a healing wound. This study introduces the concept of immune triage, in that when faced with multiple insults the immune system prioritizes responses. This paradigm likely applies to many situations that involve the innate immune system, and understanding how the innate immune system handles multiple insults is essential to understanding how it can efficiently clear pathogens while responding to other inflammatory events.

Pulmonary influenza A virus infection leads to suppression of the innate immune response to dermal injury.

Pneumonia is a world health problem and a leading cause of death, particularly affecting children and the elderly (1, 2). Bacterial pneumonia following infection with influenza A virus (IAV) is associated with increased morbidity and mortality but the mechanisms behind this phenomenon are not yet well-defined (3). Host resistance and tolerance are two processes essential for host survival during infection. Resistance is the host's ability to clear a pathogen while tolerance is the host's ability to overcome the impact of the pathogen as well as the host response to infection (4-8). Some studies have shown that IAV infection suppresses the immune response, leading to overwhelming bacterial loads (9-13). Other studies have shown that some IAV/bacterial coinfections cause alterations in tolerance mechanisms such as tissue resilience (14-16). In a recent analysis of nasopharyngeal swabs from patients hospitalized during the 2013-2014 influenza season, we have found that a significant proportion of IAV-infected patients were also colonized with , a gram-negative bacteria known to be an opportunistic pathogen in a variety of diseases (17). Mice that were infected with  following IAV infection demonstrated decreased survival and significant weight loss when compared to mice infected with either single pathogen. Using this model, we found that IAV/ coinfection of the lung is characterized by an exaggerated inflammatory immune response. We observed early inflammatory cytokine and chemokine production, which in turn resulted in massive infiltration of neutrophils and inflammatory monocytes. Despite this swift response, the pulmonary pathogen burden in coinfected mice was similar to singly-infected animals, albeit with a slight delay in bacterial clearance. In addition, during coinfection we observed a shift in pulmonary macrophages toward an inflammatory and away from a tissue reparative phenotype. Interestingly, there was only a small increase in tissue damage in coinfected lungs as compared to either single infection. Our results indicate that during pulmonary coinfection a combination of seemingly modest defects in both host resistance and tolerance may act synergistically to cause worsened outcomes for the host. Given the prevalence of  detected in human IAV patients, these dysfunctional tolerance and resistance mechanisms may play an important role in the response of patients to IAV.

Coinfection With Influenza A Virus and : An Underrecognized Impact on Host Resistance and Tolerance to Pulmonary Infections.

Neutrophil recruitment into the lung airspaces plays an important role in the containment and clearance of bacteria. Hemorrhagic shock, a complication of traumatic injury, induces immune dysfunction that compromises host defense and frequently leads to secondary infection. The objective of the current study was to determine whether prior hemorrhage impacts neutrophil recruitment in response to secondary Pseudomonas aeruginosa. Experiments were performed using a mouse model (C57BL/6) of respiratory infection by P. aeruginosa (strain PA103, 3 × 10 colony-forming units [CFUs]) that is delivered by intratracheal inhalation 24 h after hypovolemic hemorrhagic shock (fixed mean arterial blood pressure at 35 mmHg for 90 min, Ringer's lactate infused as fluid resuscitation). By postmortem flow cytometry analyses of bronchoalveolar lavage fluid, we observe that prior hemorrhage attenuates the entry of neutrophils into the lung airspaces in response to P. aeruginosa. The reduction in neutrophil recruitment occurs in an amplified inflammatory environment, with elevated lung tissue levels of interleukin 6 and C-X-C motif ligand 1 in mice receiving hemorrhage prior to infection. As compared to either insult alone, outcome to sequential hemorrhage and respiratory infection includes enhanced mortality. The effect of prior hemorrhage on clearance of P. aeruginosa, as determined by quantifying bacterial CFUs in lung tissue, was not statistically significant at 24 h postinfection, but our data suggest that further inquiry may be needed to fully understand the potential impact of hemorrhagic shock on this process. These results suggest that changes in neutrophil recruitment may contribute to the immune dysfunction following hemorrhagic shock that renders the host susceptible to severe respiratory infection.

Article	Year
Role of tissue protection in lethal respiratory viral-bacterial coinfection. Science (New York, N.Y.)\| PubMed ID: 23618765	2013
The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature\| PubMed ID: 24553136	2014
Influence of the microbiome on response to vaccination. Human vaccines & immunotherapeutics\| PubMed ID: 26090701	2015
Type I Interferon Signaling Prevents IL-1β-Driven Lethal Systemic Hyperinflammation during Invasive Bacterial Infection of Soft Tissue. Cell host & microbe\| PubMed ID: 26962946	2016
The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes. PLoS pathogens\| PubMed ID: 27973535	2016
Surviving Deadly Lung Infections: Innate Host Tolerance Mechanisms in the Pulmonary System. Frontiers in immunology\| PubMed ID: 29988424	2018
Pulmonary influenza A virus infection leads to suppression of the innate immune response to dermal injury. PLoS pathogens\| PubMed ID: 30138446	2018
Coinfection With Influenza A Virus and : An Underrecognized Impact on Host Resistance and Tolerance to Pulmonary Infections. Frontiers in immunology\| PubMed ID: 30420852	2018
Hemorrhage Attenuates Neutrophil Recruitment in Response to Secondary Respiratory Infection by Pseudomonas Aeruginosa. Shock (Augusta, Ga.)\| PubMed ID: 30475329	2019

Amanda M. Jamieson

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