Erin E. Perrone

Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus clearance

BACKGROUND Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare. MATERIALS AND METHODS We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia. RESULTS Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P < 0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P < 0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight. CONCLUSIONS These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

Cancer causes increased mortality and is associated with altered apoptosis in murine sepsis

Objective:Whereas most septic patients have an underlying comorbidity, most animal models of sepsis use mice that were healthy before the onset of infection. Malignancy is the most common comorbidity associated with sepsis. The purpose of this study was to determine whether mice with cancer have a different response to sepsis than healthy animals. Design:Prospective, randomized controlled study. Setting:Animal laboratory in a university medical center. Subjects:C57Bl/6 mice. Interventions:Animals received a subcutaneous injection of either 250,000 cells of the transplantable pancreatic adenocarcinoma cell line Pan02 (cancer) or phosphate-buffered saline (healthy). Three weeks later, mice given Pan02 cells had reproducible, nonmetastatic tumors. Both groups of mice then underwent intratracheal injection of either Pseudomonas aeruginosa (septic) or 0.9% NaCl (sham). Animals were killed 24 hrs postoperatively or followed-up 7 days for survival. Measurements and Main Results:Mice with cancer and healthy mice appeared similar when subjected to sham operation, although cancer animals had lower levels of T- and B-lymphocyte apoptosis. Septic mice with cancer had increased mortality compared to previously healthy septic mice subjected to the identical injury (52% vs. 28%; p = .04). This was associated with increased bacteremia but no difference in local pulmonary infection. Septic mice with cancer also had increased intestinal epithelial apoptosis. Although sepsis induced an increase in T- and B-lymphocyte apoptosis in all animals, septic mice with cancer had decreased T- and B-lymphocyte apoptosis compared to previously healthy septic mice. Serum and pulmonary cytokines, lung histology, complete blood counts, and intestinal proliferation were similar between septic mice with cancer and previously healthy septic mice. Conclusions:When subjected to the same septic insult, mice with cancer have increased mortality compared to previously healthy animals. Decreased systemic bacterial clearance and alterations in intestinal epithelial and lymphocyte apoptosis may help explain this differential response.

Intestinal resection induces angiogenesis within adapting intestinal villi

PURPOSE Adaptive growth of the intestinal mucosa in response to massive gut loss is fundamental for autonomy from parenteral nutrition. Although angiogenesis is essential for cellular proliferation in other tissues, its relevance to intestinal adaptation is unknown. We tested the hypothesis that resection-induced adaptation is associated with new blood vessel growth. METHODS Male C57Bl/6 mice underwent either a 50% small bowel resection or a sham (transection and reanastomosis) operation. After 1, 3, or 7 days, capillary density within the intestinal villi was measured using confocal microscopy. A messenger RNA reverse-transcriptase polymerase chain reaction (RT-PCR) array was used to determine angiogenic gene expression during adaptation. RESULTS Mice that underwent small bowel resection had a significantly increased capillary density compared to sham-operated mice at postoperative day 7. This morphological alteration was preceded by significant alterations in 5 candidate genes at postoperative day 3. CONCLUSION New vessel blood growth is observed in the adapting intestine after massive small bowel loss. This response appears to follow rather than initiate the adaptive alterations in mucosal morphology that are characteristic of adaptation. A better understanding of this progress and the signaling factors involved may improve therapeutic options for children with short gut syndrome.

Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia–induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus–induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid−/− mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand−/− mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

Cecal ligation and puncture followed by methicillin-resistant Staphylococcus aureus pneumonia increases mortality in mice and blunts production of local and systemic cytokines.

ABSTRACT Mortality in the intensive care unit frequently results from the synergistic effect of two temporally distinct infections. This study examined the pathophysiology of a new model of intra-abdominal sepsis followed by methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Mice underwent cecal ligation and puncture (CLP) or sham laparotomy followed 3 days later by an intratracheal injection of MRSA or saline. Both CLP/saline and sham/MRSA mice had 100% survival, whereas animals with CLP followed by MRSA pneumonia had 67% 7-day survival. Animals subjected to CLP/MRSA had increased bronchoalveolar lavage concentrations of MRSA compared with sham/MRSA animals. Animals subjected to sham/MRSA pneumonia had increased bronchoalveolar lavage levels of interleukin 6 (IL-6), tumor necrosis factor &agr;, and granulocyte colony-stimulating factor compared with those given intratracheal saline, whereas CLP/MRSA mice had a blunted local inflammatory response with markedly decreased cytokine levels. Similarly, animals subjected to CLP/saline had increased peritoneal lavage levels of IL-6 and IL-1&bgr; compared with those subjected to sham laparotomy, whereas this response was blunted in CLP/MRSA mice. Systemic cytokines were upregulated in both CLP/saline and sham/MRSA mice, and this was blunted by the combination of CLP/MRSA. In contrast, no synergistic effect on pneumonia severity, white blood cell count, or lymphocyte apoptosis was identified in CLP/MRSA mice compared with animals with either insult in isolation. These results indicate that a clinically relevant model of CLP followed by MRSA pneumonia causes higher mortality than could have been predicted from studying either infection in isolation, and this was associated with a blunted local (pulmonary and peritoneal) and systemic inflammatory response and decreased ability to clear infection.

Inhibition of Intestinal Epithelial Apoptosis Improves Survival in a Murine Model of Radiation Combined Injury

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

p21waf1/cip1 deficiency does not perturb the intestinal crypt stem cell population after massive small bowel resection

BACKGROUND After small bowel resection (SBR), adaptation is initiated in intestinal crypts where stem cells reside. Prior studies revealed SBR-induced enterocyte proliferation requires the expression of p21(waf1/cip1). As deficient expression of p21(waf1/cip1) has been shown to result in reduced numbers of hematopoietic stem cells. We sought to test the hypothesis that p21(waf1/cip1)deficiency similarly perturbs the intestinal stem cell population after SBR. METHODS Control (n = 21; C57Bl/6) and p21(waf1/cip1)-null mice (n = 30) underwent 50% proximal SBR or sham operation. After 3 days, the ileum was harvested and the crypt stem cell population evaluated by counting crypt base columnar cells on histologic sections, determining the expression of Musashi-1 and Lgr5, and profiling the transcriptional expression of 84 known stem cell genes. RESULTS There were no significant differences in crypt base columnar cells, expression of Musashi-1 or Lgr5, or in stem cell gene expression after SBR in control mice. Furthermore, there were no differences in these markers between controls and p21(waf1/cip1)-null mice. CONCLUSION In contrast with bone marrow stem cells, the stem cell population of the gut is unaffected by deficient expression of p21(waf1/cip1). Additional mechanisms for the role of p21(waf1/cip1) in small bowel proliferation and adaptation after massive SBR must be considered.