Establishing sub-phenotypes of pneumonia based on distinct host processes will be a step towards using host-directed therapies (to complement microbe-directed therapies) more rationally and precisely. Although pneumonia is a pulmonary pathophysiology, histological changes within the lungs have not been leveraged for sub-phenotyping. We addressed this by scoring 18 histopathology features (e.g., type 2 cell hyperplasia or necrosis) across rapid autopsy lung samples from 276 elderly subjects with pneumonia. Machine learning algorithms segregated subjects into seven different sub-phenotypes of pneumonia with distinct histopathology signatures. Quantitative immunofluorescence demonstrated associations of macrophages, neutrophils, T cells, and B cells with select histology features and pulmonary pathology sub-phenotypes. Mouse models revealed corollary sub-phenotypes, although some histology features observed in human lungs were never observed in mice. By illuminating this spectrum of histopathologies and discriminating discrete sub-phenotypes of pneumonia, a foundational framework emerges for developing and using host-directed therapies for subsets of pneumonia patients.

Abstract Introduction Pulmonary infections induce heterogeneous lung immune responses, resulting in multiple pneumonia histopathology sub-phenotypes. Necrosuppurative pneumonia is characterized by alveolar necrosis, neutrophils, edema, and airspace fibrin accumulation, observed in human autopsies and S. pneumoniae (Sp)-infected mice. We endeavor to elucidate innate immunity mechanisms leading to the necrosuppurative sub-phenotype of pneumonia. Methods We compared outcomes of Sp pneumonia in wild-type (WT) mice and those with tissue factor (TF) inducibly deleted from lung epithelial cells. Results In WT mice, fibrinogen mRNA was induced in the liver and elevated in the blood during pneumonia, while TF mRNA was strongest in lung epithelial cells. Bacteria grew inexorably in the lungs and caused bacteremia, which was not affected by deletion of TF from the lung epithelium. However, deletion of lung epithelial cell TF reduced alveolar fibrin deposition and neutrophil recruitment. While Sp infection caused alveolar necrosis in WT mice, we instead observed type II epithelial cell hyperplasia when TF was deleted from the lung epithelium. Conclusion The airspace fibrin, neutrophil accumulation, and epithelial necrosis that are defining features of necrosuppurative pneumonia caused by severe pneumococcal infection all depend on TF produced by lung epithelial cells. Funding Source NIH-NHLBI Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Pulmonary infections induce heterogeneous immune responses in the lung, resulting in multiple pneumonia phenotypes. An etiological agent cannot be identified in the majority of pneumonia cases; thus, elucidating these heterogeneous lung pathobiologies for the development of host-directed therapies is a major research priority. To characterize the heterogeneity in human pneumonia pathobiology, we scored 20 pneumonia features across hundreds of autopsy tissue samples from elderly subjects who died with pneumonia. These pneumonia features varied significantly across our human lung samples, particularly the presence and severity of polymerized fibrin in the alveolar spaces. In our human pneumonia samples, alveolar fibrin deposition was most severe in the samples diagnosed with bronchopneumonia and positively-correlated with neutrophilia and necrosis, while it was less frequent in our samples diagnosed with interstitial pneumonia and negatively-correlated with lymphoplasmacytosis and fibrosis. To understand the mechanism and significance of alveolar fibrin deposition during pneumonia, we further characterized these features in C57BL/6 mice with severe pneumonias caused by Streptococcus pneumoniae (Sp), Escherichia coli (Ec), Klebsiella pneumoniae (Kp), influenza A virus (IAV), or SARS-CoV-2 (SCV2). Sp- and Ec-infected lungs were dominated by neutrophilic influx and high levels of polymerized fibrin in the alveolar spaces, reflecting the fibrin-neutrophil association observed in human autopsy samples, while Kp-, IAV-, and SCV2-infected lungs had little-to-no alveolar fibrin staining despite an abundance of fibrin in the vasculature. During Sp infection, fibrinogen was upregulated in the liver and significantly increased in the blood. RNA in situ hybridization of Sp-infected lungs revealed an upregulation of extrinsic coagulation factors that promote fibrin polymerization, including tissue factor (F3) in airway epithelial cells, F10 in recruited neutrophils, and F13a1 in myeloid cells, revealing potential mechanistic drivers of fibrin polymerization in the airspace. Together, these data demonstrate that (1) alveolar fibrin is observed in only a subset of human lungs with pneumonia, and (2) some pneumonia-causing pathogens, but not others, upregulate fibrinogen and promote cleaved fibrin polymerization in the alveolar spaces of mice. These mouse models can be used to elucidate the immunological and pathophysiological significance of this fibrin accumulation in a subset of pneumonias, which may provide additional targets for the development of host-directed therapies aiming to enhance or reduce alveolar fibrin deposition during pneumonia.

ABSTRACT:Shock® publishes over 200 scientific papers each year related to the field of shock, including trauma and sepsis. While these excellent papers report objective data about these conditions, they do not provide clinical stories that humanize the disorders or provide the surviving patients' perspective. This Commentary presents two patient stories that illustrate the relevance of patient perception in the context of research published in Shock®. The intent is to link compelling, memorable stories that highlight how recent scientific literature addresses clinical issues. Such an additional perspective may be used by the Shock readers to communicate more effectively with non-medical policy makers and the lay public to effectively justify funding for shock research.

Rationale: Pneumonia induces heterogeneous immune and pathophysiological processes in the lung. We aimed to better define these responses using pulmonary histopathology, and to determine whether varying pulmonary pathologies yielded distinct subphenotypes of pneumonia. Mouse models are often used to study human pneumonias, yet we lack a direct comparison between the two. We aimed to characterize the similarities and disconnects between mouse and human pneumonia. Methods: H&E slides from elderly subjects with pneumonia diagnosis at autopsy n=276) and with no known lung disease (n=16) were scored across 18 histopathological features (e.g., necrosis, hyaline membranes, peribronchiolar metaplasia, etc.) by 2 pathologists. Multiplex fluorescence immunohistochemistry (mfIHC) was used to quantify 5 immune cell types including CD4+ T helper cells, CD8+ cytotoxic T cells, MPO+ neutrophils, CD68+ macrophages, and CD19+ B cells on a subset of samples (n=159). 100 mouse samples were scored for the same histopathological features across multiple models including bacterial (pneumococcus serotypes 3 or 19F, E.coli, or Klebsiella pneumoniae) and viral (Influenza A virus PR8, and 2 mouse-adapted strains of SARS-CoV2) infections of young adult and (for select pathogens) elderly mice. We used a machine learning (ML) framework to characterize subphenotypes in humans and mice. Results: Each histopathology feature demonstrated variation across pneumonic human subjects, and no single feature or set of features consistently defined pneumonia. Our ML framework segregated samples into 7 clusters of human pneumonias with distinct histopathological patterns (e.g., 3.6% had coincidently high type 2 cell hyperplasia, hyaline membranes, lymphoplasmacytosis, and fibrosis, while a different 19% were distinguished by high neutrophils, edema, alveolar fibrin, and necrosis without hemorrhage). mfIHC indicated that immune cells associated with specific features and clusters (e.g., CD68+ cells positively associated with the alveolar hemorrhage feature and with the cluster of 11% of samples for which that feature had the highest variable importance score). The mouse samples also segregated into distinct pulmonary pathology clusters. Combined clustering of human & mice samples, averaged per cluster indicated that each mouse cluster could be linked to corresponding human clusters. However, some histopathology features observed in humans (e.g., abscess & granuloma) were never observed in these mouse models. Conclusions: Our analyses indicate seven distinct pulmonary pathology sub-phenotypes of pneumonia. Refinements to mouse models are needed to fully capture the diverse histopathological landscape of human pneumonias, but the mouse models examined will have promising utility for assessing the biology of pneumonia pulmonary pathology sub-phenotypes including underlying mechanisms and pathophysiological significance.

Asthma is a chronic condition with high morbidity and healthcare costs, and cockroach allergens are an established cause of urban pediatric asthma. A better understanding of cell types involved in promoting lung inflammation could provide new targets for the treatment of chronic pulmonary disease. Because of its role in regulating myeloid cell-dependent inflammatory processes, we examined A2B R expression by myeloid cells in a cockroach allergen model of murine asthma-like pulmonary inflammation. Both systemic and myeloid tissue-specific A2B R deletion significantly decreased pulmonary inflammatory cell recruitment, airway mucin production, and proinflammatory cytokine secretion after final allergen challenge in sensitized mice. A2B R deficiency resulted in a dramatic reduction on Th2-type airways responses with decreased pulmonary eosinophilia without augmenting neutrophilia, and decreased lung IL-4, IL-5, and IL-13 production. Chemokine analysis demonstrated that eotaxin 1 and 2 secretion in response to repeated allergen challenge is myeloid cell A2B R dependent. In contrast, there were no differences in the levels of the CXC chemokines keratinocyte-derived chemokine and MIP-2 in the myeloid cell A2B R-deficient mice, strengthening A2B R involvement in the development of Th2-type airways inflammation. Proinflammatory TNF-α, IFN-γ, and IL-17 secretion were also reduced in systemic and myeloid tissue-specific A2B R deletion mouse lines. Our results demonstrate Th2-type predominance for A2B R expression by myeloid cells as a mechanism of development of asthma-like pulmonary inflammation.

ABSTRACT:Background: Sepsis accounts for substantial morbidity and mortality motivating investigators to continue the search for pathways and molecules driving the pathogenesis of the disease. The current study examined if the novel C-type lectin receptor (CLR), Clec2d, plays a significant role in the pathogenesis of sepsis. Methods: Clec2d knockout (KO) mice were fully backcrossed onto the C57/BL6 background. Acute endotoxemia was induced with an intraperitoneal injection of lipopolysaccharide (LPS). Sepsis was induced in two different models, cecal ligation and puncture (CLP) and Pseudomonas aeruginosa pneumonia. Both models were treated with antibiotics and fluid resuscitation. In the sepsis models, physiologic and hematologic measurements were measured at 24 h by collecting a small sample of peripheral blood. Mortality was followed for 14 days. Results : A total of 197 mice were studied, 58 wild type (WT) and 54 knock-out (KO) in the LPS model; 27 wild type and 21 KO mice in the CLP model; and 22 WT and 15 KO mice in the pneumonia model. Clec2d KO mice had greater mortality in the LPS and CLP studies but not the pneumonia model. There were significant differences in multiple parameters determined 24 h post sepsis between mice who subsequently died and those lived. Consistent with previous reports in the CLP model, higher concentrations of IL-6, increased numbers of peripheral blood lymphocytes and greater renal injury were found in the dying mice. In contrast, in the pneumonia model, IL-6 was higher in the surviving mice; however, the IL-6 levels in the pneumonia model (0.6 ± 0.3 ng/mL mean ± SEM) were less than 2% of the IL-6 levels of mice that died in the CLP model (41 ± 9 ng/mL, mean ± SEM). There were no differences in the lymphocyte count or renal injury between living and dying mice in the pneumonia model. In both sepsis models, dying mice had lower heart rates, respiratory rates, and body temperatures. These values were also lower in the KO mice compared to the WT in CLP, but the breath rate and body temperature were increased in the KO pneumonia mice. Conclusion: The C-type lectin receptor Clec2d plays a complicated role in the pathogenesis of sepsis, which varies with source of infection as demonstrated in the models used to study the disease. These data highlight the heterogeneity of the responses to sepsis and provide further evidence that a single common pathway driving sepsis organ injury and death likely does not exist.

Artificial intelligence (AI) may have a profound impact on traditional teaching in academic settings. Multiple concerns have been raised, especially related to using ChatGPT for creating de novo essays. However, AI programs such as ChatGPT may augment teaching techniques. In this article, we used ChatGPT 3.5 to create 60 multiple choice questions. Author written text was uploaded and ChatGPT asked to create multiple choice questions with an explanation for the correct answer and explanations for the incorrect answers. Unfortunately, ChatGPT only generated correct questions and answers with explanations in 32 % of the questions (19 out of 60). In many instances, ChatGPT failed to provide an explanation for the incorrect answers. An additional 25 % of the questions had answers that were either wrong or misleading. A grade of 32 % would be considered failing in most courses. Despite these issues, instructors may still find ChatGPT useful for creating practice exams with explanations-with the caveat that extensive editing may be required.

BackgroundThe creatinine height index (CHI) is an estimate of lean body mass. We hypothesize that a modified CHI estimate using serum creatinine (sCr) levels in patients with normal renal function when performed soon after injury would reflect preinjury protein nutrition status. MethodsThe urine CHI (uCHI) was calculated using the 24-h urine sample. The serum-derived estimated CHI (sCHI) was calculated using the sCr on admission. Correlation between abdominal computed tomography images at specific lumbar vertebral levels and total body fat and muscle content was used for comparison as an independent measurement of nutrition status unlikely to be substantially altered by trauma. ResultsA total of 45 patients were enrolled, all with a significant injury burden (median injury severity score [ISS] = 25; interquartile range, 17-35). The calculated sCHI on admission was 71.0% (SD = 26.9%) and likely underestimates the CHI when compared with uCHI (mean = 112.5%, SD = 32.6%). Stratifying by degree of stress demonstrated that in a group of 23 moderately and severely stressed patients, uCHI (mean = 112.7%, SD = 5.7%) and sCHI (mean = 60.8%, SD = 1.9%) were significantly different and without correlation (r = -0.26, P = 0.91). In patients without stress, there was a significant negative correlation between sCHI and psoas muscle area (r = -0.869, P = 0.03), and in patients with severe stress there was a significant positive correlation between uCHI and psoas muscle area (r = 0.733, P = 0.016). ConclusionThe CHI calculated from the initial sCr is not an appropriate estimate of uCHI in critically ill trauma patients and is not a valid measure of psoas muscle mass in this setting.

Remick, Daniel; Szabó, Andrea; Juffermans, Nicole; Osuchowski, Marcin F. Author Information

ABSTRACT:Background: Acute kidney injury (AKI) occurs frequently in septic patients and correlates with increased mortality. Because clinical studies investigating hydrocortisone, ascorbic acid, and thiamine (HAT) have demonstrated discordant results, studies were performed using mortality stratification for therapy to identify candidates for therapy and determine mechanisms of organ injury. Methods: Sepsis was induced using the cecal ligation and puncture (CLP) model of sepsis with fluid and antibiotic support. Heart rate (HR) measurements obtained 6 hours after CLP stratified mice into live predicted (P-Live) or die predicted (P-Die). Stratified mice were then randomized for treatment with HAT or vehicle given 7 hours after CLP. Physiologic measurements were taken again at 24 hours, and mice were killed to collect blood and organs. Results: The following five groups were created: (1) P-Live vehicle, (2) P-Live HAT, (3) P-Die vehicle, (4) P-Die HAT, and (5) naive mice. Comparisons were made to test the hypotheses that (1) P-Die vehicle mice will have significant deterioration compared with P-Live mice targeting the kidney and (2) HAT will correct these deleterious changes in P-Die mice. Compared with P-Live, P-Die mice had a significant decline in all measured physiologic parameters (HR, cardiac output, breath rate, and temperature), which were corrected with HAT therapy (P < 0.05 for all parameters). The P-Die mice had declines in the ascorbic acid within the blood, peritoneal lavage, and kidney homogenate compared with P-Live mice indicating consumption, and the decline was corrected with HAT. Elevated IL-6, KC, Macrophage Inflammatory Protein-2, and IL-1RA were found in P-Die mice and decreased with HAT. Markers of endothelial cell injury (glypican 1 and glypican 4) were elevated in the P-Die mice, and these values were decreased with HAT therapy. Low oxygen levels with subsequent oxidative stress (OS) in the kidney were visualized in histologic sections using hypoxyprobe and also with carbonyl proteins and 8-iso-prostaglandin F2α in kidney homogenates. The P-Die mice had significant elevations of renal OSs, which was ameliorated with HAT. Kidney injury was evident in the P-Die mice compared with P-Live mice with elevations in blood urea nitrogen and cystatin C, which were significantly reduced with HAT. There was no evidence of global hypoxia or organ injury because hepatic parameters remained normal. Conclusions: Our data show that in CLP-induced sepsis, P-Die mice have increased inflammation, OS, and kidney injury. Hydrocortisone, ascorbic acid, and thiamine therapy decreased renal OS and injury in the P-Die group when given after the onset of sepsis-induced physiologic changes.

BACKGROUND: There is a lack of consensus regarding the optimal nutritional support for trauma patients. We hypothesize that early postinjury metabolic support focusing on adequate protein would modify the metabolic signature and alter the inflammatory environment for critically ill trauma patients. METHODS: We conducted a prospective randomized controlled pilot trial for adult patients admitted to the surgical intensive care unit following traumatic injury. Patients were randomized to receive early metabolic support (EMS) (peripheral amino acid infusions) or standard of care (enteral nutrition as soon as feasible). Routine laboratory assessments, nitrogen balance, cytokines, and metabolomic analyses were assessed at baseline and day 5 after intervention. RESULTS: A total of 42 trauma patients were randomized into well-balanced groups with similar age (32 years), Injury Severity Score (25), and body mass index (27.4 kg/m(2)). Early metabolic support provided significantly more protein (1.43 g/kg vs. 0.35 g/kg; p < 0.0001) and more calories (12.6 kcal/kg vs. 7.5 g/kg; p = 0.0012) over the first 5 days as compared with the standard of care. Early metabolic support modified protein catabolism and synthesis as demonstrated by a larger median negative nitrogen balance (-16.3 g vs. -5.3 g; p = 0.03) and a unique metabolomic profile at day 5. The biochemical profile of patients who received EMS was defined by greater declines in circulating levels of stress hormone precursors and increased levels of amino acids. The inflammatory response following EMS resulted in a greater decrease in interleukin-1B (p = 0.02) and increase in soluble interleukin-6 receptor (p = 0.01) between baseline and day 5 as compared with the standard of care. The EMS group had a decreased length of stay (15 vs. 22 days) and decreased surgical intensive care unit length of stay (8 vs. 9 days); however, this disappeared after adjustment for Injury Severity Score in this small population. CONCLUSIONS: Early metabolic support with amino acid is safe, modifies metabolism, and may downregulate the inflammatory state associated with significant trauma, warranting a larger trial to assess for improved outcomes.

BACKGROUND Multiple clinical trials failed to demonstrate the efficacy of hydrocortisone, ascorbic acid, and thiamine (HAT) in sepsis. These trials were dominated by patients with pulmonary sepsis and have not accounted for differences in the inflammatory responses across varying etiologies of injury/illness. Hydrocortisone, ascorbic acid, and thiamine have previously revealed tremendous benefits in animal peritonitis sepsis models (cecal ligation and puncture [CLP]) in contradiction to the various clinical trials. The impact of HAT remains unclear in pulmonary sepsis. Our objective was to investigate the impact of HAT in pneumonia, consistent with the predominate etiology in the discordant clinical trials. We hypothesized that, in a pulmonary sepsis model, HAT would act synergistically to reduce end-organ dysfunction by the altering the inflammatory response, in a unique manner compared with CLP. METHODS Using Pseudomonas aeruginosa pneumonia, a pulmonary sepsis model (pneumonia [PNA]) was compared directly to previously investigated intra-abdominal sepsis models. Machine learning applied to early vital signs stratified animals into those predicted to die (pDie) versus predicted to live (pLive). Animals were then randomized to receive antibiotics and fluids (vehicle [VEH]) vs. HAT). Vitals, cytokines, vitamin C, and markers of liver and kidney function were assessed in the blood, bronchoalveolar lavage, and organ homogenates. RESULTS PNA was induced in 119 outbred wild-type Institute of Cancer Research mice (predicted mortality approximately 50%) similar to CLP. In PNA, interleukin 1 receptor antagonist in 72-hour bronchoalveolar lavage was lower with HAT (2.36 ng/mL) compared with VEH (4.88 ng/mL; p = 0.04). The remaining inflammatory cytokines and markers of liver/renal function showed no significant difference with HAT in PNA. PNA vitamin C levels were 0.62 mg/dL (pDie HAT), lower than vitamin C levels after CLP (1.195 mg/dL). Unlike CLP, PNA mice did not develop acute kidney injury (blood urea nitrogen: pDie, 33.5 mg/dL vs. pLive, 27.6 mg/dL; p = 0.17). Furthermore, following PNA, HAT did not significantly reduce microscopic renal oxidative stress (mean gray area: pDie, 16.64 vs. pLive, 6.88; p = 0.93). Unlike CLP where HAT demonstrated a survival benefit, HAT had no impact on survival in PNA. CONCLUSION Hydrocortisone, ascorbic acid, and thiamine therapy has minimal benefits in pneumonia. The inflammatory response induced by pulmonary sepsis is unique compared with the response during intra-abdominal sepsis. Consequently, different etiologies of sepsis respond differently to HAT therapy.

Biomarkers have been used in sepsis to assist with the diagnosis of disease as well as determining the severity of disease, that is, prognosis. These biomarkers are based on the presence of discrete molecules within the blood. Unfortunately, in 2020, a single biomarker does not have sufficient sensitivity and specificity to definitively rule in or rule out sepsis. Biomarkers have shown better performance in animal models of disease.

Supplemental Digital Content is available in the text ABSTRACT Background: “Cytokine storm” has been used to implicate increased cytokine levels in the pathogenesis of serious clinical conditions. Similarities with Severe Acute Respiratory Syndrome Coronoavirus-2 (SARS CoV-2) and the 2012 Middle Eastern Respiratory Syndrome led early investigators to suspect a “cytokine storm” resulting in an unregulated inflammatory response associated with the significant morbidity and mortality induced by SARS CoV-2. The threshold of blood cytokines necessary to qualify as a “cytokine storm” has yet to be defined. Methods: A literature review was conducted to identify cytokine levels released during 11 assorted clinical conditions or diseases. Weighted averages for various cytokines were calculated by multiplying the number of patients in the paper by the average concentration of each cytokine. Correlation between cytokine levels for individual conditions or diseases were assessed using Pearson correlation coefficient. Results: The literature was reviewed to determine blood levels of cytokines in a wide variety of clinical conditions. These conditions ranged from exercise and autoimmune disease to septic shock and therapy with chimeric antigen receptor T cells. The most frequently measured cytokine was IL-6 which ranged from 24,123 pg/mL in septic shock to 11 pg/mL after exercise. In patients with severe SARS CoV-2 infections, blood levels of IL-6 were only 43 pg/mL, nearly three magnitudes lower than IL-6 levels in patients with septic shock. The clinical presentations of these different diseases do not correlate with blood levels of cytokines. Additionally, there is poor correlation between the concentrations of different cytokines among the different diseases. Specifically, blood levels of IL-6 did not correlate with levels of IL-8, IL-10, or TNF. Septic shock had the highest concentrations of cytokines, yet multiple cytokine inhibitors have failed to demonstrate improved outcomes in multiple clinical trials. Patients with autoimmune diseases have very low blood levels of cytokines (rheumatoid arthritis, IL-6 = 34 pg/mL; Crohn's disease, IL-6 = 5 pg/mL), yet respond dramatically to cytokine inhibitors. Conclusion: The misleading term “cytokine storm” implies increased blood levels of cytokines are responsible for a grave clinical condition. Not all inflammatory conditions resulting in worsened disease states are correlated with significantly elevated cytokine levels, despite an association with the term “cytokine storm”. “Cytokine storm” should be removed from the medical lexicon since it does not reflect the mediators driving the disease nor does it predict which diseases will respond to cytokine inhibitors.

Neutrophils play a critical role in the eradication of pathogenic organisms, particularly bacteria. However, in the septic patient the prolonged activation and accumulation of neutrophils may augment tissue and organ injury. This review discusses the different activation states and chemotaxis of neutrophils in septic patients. Neutrophil killing of bacteria and the formation of neutrophil extracellular traps represent important components of the innate immune response and they become dysregulated during sepsis, possibly through changes in their metabolism. Delayed neutrophil apoptosis may contribute to organ injury, or allow better clearance of pathogens. Neutrophils provide a friendly immune response to clear infections, but excessive activation and recruitment has the potential to turn them into potent foes.

Reply: We appreciate the insightful comments on the above Letter to the Editor, which discusses how our findings (similar to our previously published work on the topic) (1, 2) may contradict those of prior studies on Trichostatin A (TSA) inhibition of the Histone Deacetylase (HDAC) complex (3). We also want to thank the Editor for giving us the opportunity to respond to the Letter. Citing a research letter by Finnin et al. (4) one of our manuscript readers posits that TSA exerts its HDAC inhibitory effect by directly binding to the HDAC catalytic region, and hence should not have any effect on HDAC7 expression. However, the cited manuscript merely assessed the TSA ability to bind to a bacterial HDAC homologue (a protein that shares only 35.2% of the amino acid sequence of human and murine HDAC1, as clearly pointed out in the referenced manuscript) in vitro, not its effect on HDAC expression. Therefore, an argument that TSA does not alter HDAC7 expression cannot be made from the referenced article, as this topic is not even explored in the cited manuscript. The reader also states that there are no papers to his knowledge describing the effect of TSA on HDAC expression. Not only are there too numerous to cite in a Scientific Letter manuscripts that have studied HDAC expression after TSA administration, but one of the more prominent ones is cited by the reader himself: In that article, Yagi et al. (5) demonstrate that HDAC9 and HDAC11 expression decrease after TSA administration. That HDAC11 is downregulated with TSA in the Yagi manuscript, is consistent with our findings, despite the different models of Acute Lung Injury used between Yagi's manuscript (two-hit viral and bacterial model) and ours (gram-negative bacterial pneumonia), and the different timepoints (36 h in the Yagi manuscript vs. 6 h in ours) selected for assessment. The reader states that we claim that the “E coli effects is due, at least in part, to the changes in HDAC7 expression,” while a closer examination of our manuscript reveals no such claim. It is also stated that the effects of HDAC7 siRNA and TSA cannot be found on HDAC7 expression, while those are clearly demonstrated, not only for HDAC7, but for all 1-11 HDACS and Sirt1 in Figure 4. We want to thank the author for pointing out a typographical error that we inadvertently made, however. The statement “This transcription is inhibited further with both TSA and highly selective HDAC7-siRNA inhibition” should read “This transcription is inhibited further with TSA administration.” The reader also “assumes that the authors used an excess of siRNA to efficiently inhibit HDAC7 production” to refute our hypothesis of HDAC7 mRNA restoration due to rapid turnover, while we clearly state that “we demonstrated >40% inhibition of quantifiable HDAC7 protein with HDAC7-siRNA” (Fig. 5). We also state in the Discussion that we were satisfied with this low-level HDAC7 inhibition in that this effect may be more clinically relevant and simulating real-life pharmacologic inhibition, as opposed to near zero protein levels. A statement that “any siRNA designed to decrease expression of specific mRNA, but not protein, therefore, it is unclear, how the protein level of HDAC7 decreased without decreasing HDAC7 mRNA level” is irrelevant to our project, as we specifically aimed to limit HDAC7 protein translation, as this is the enzymatically active form of HDAC7 (mRNA is an enzymatically inactive nucleotide, functioning merely as a guide for the HDAC7 protein synthesis). We also propose a hypothesis for this finding, which we are currently actively testing. We agree that the finding of HDAC7 mRNA and protein levels moving in opposite directions in the face of infection is highly interesting, just like our reader, and propose potential mechanisms for this, and aim to study this phenomenon further in the near future. Lastly, the reader observes that with TSA and HDAC7 siRNA not having the same effects on both mRNA AND protein synthesis, the conclusion that TSA acts mainly through HDAC7 inhibition cannot be made. We also want to clarify that nowhere in the manuscript is such a claim made, rather HDAC7 inhibition is key to the regulation of the early inflammatory response that leads to Acute Lung Injury after gram-negative pneumonia. It is also important to reiterate that only the protein is believed to be enzymatically active in our clinical relevant scenario, with the mRNA merely encoding for it, and its direction of regulation is likely irrelevant, as long as the protein levels expressed remain the same.