Sepsis induces early alterations in innate immunity that impact mortality to secondary infection

Abstract

Sepsis, the systemic inflammatory response to microbial infection, induces changes in both innate and adaptive immunity that presumably lead to increased susceptibility to secondary infections, multiorgan failure, and death. Using a model of murine polymicrobial sepsis whose severity approximates human sepsis, we examined outcomes and defined requirements for survival after secondary Pseudomonas aeruginosa pneumonia or disseminated Listeria monocytogenes infection. We demonstrate that early after sepsis neutrophil numbers and function are decreased, whereas monocyte recruitment through the CCR2/MCP-1 pathway and function are enhanced. Consequently, lethality to Pseudomonas pneumonia is increased early but not late after induction of sepsis. In contrast, lethality to listeriosis, whose eradication is dependent upon monocyte/macrophage phagocytosis, is actually decreased both early and late after sepsis. Adaptive immunity plays little role in these secondary infectious responses. This study demonstrates that sepsis promotes selective early, impaired innate immune responses, primarily in neutrophils, that lead to a pathogen-specific, increased susceptibility to secondary infections.

Figure 1. Survival and bacterial colonization to intranasal Pseudomonas aeruginosa infection in post-septic mice

On days three (Panel A) and seven (Panel B) post-CLP, mice were challenged with an intranasal administration of 10⁸ cfu of Pseudomonas aeruginosa (□ healthy control animals, ▲ sham-treated animals, ■ CLP-treated animals). Survival was evaluated over the next nine days. Additional animals were sacrificed 24 hours after Pseudomonas pneumonia and bacterial colony counts in the bronchoalveolar lavage (BAL) fluid were determined (Panels C and D). Survival studies represent the outcome of n=20 animals per group, while BAL fluids represent n=7 per group. Each experiment was repeated at least three times. Significant differences in survival were determined by Fisher’s exact test, and in cfu, by ANOVA and multiple range test * = p<0.01.

Figure 2. Blood, BAL, peritoneal lavage and bone marrow neutrophil responses to polymicrobial sepsis

At intervals after polymicrobial sepsis, mice were sacrificed and blood, BAL and bone marrow neutrophils (CD11b⁺,GR-1⁺) were determined by flow cytometry Panels A and B present total BAL neutrophil numbers while Panels C-G represent peritoneal lavage, blood and bone marrow neutrophil percentages and absolute numbers. Within three to five days, there was a relative rapid influx of neutrophils into the peritoneal cavity associated with deficiency in blood, BAL and bone marrow neutrophils that returned to baseline or above by seven days after sepsis. Values represent the mean of five to seven animals per group. Each experiment was confirmed at least three times. Significance between naïve, sham and CLP at day 3 and 7 was determined by one way ANOVA. Significance between sham and CLP over time was determined by two way ANOVA. * = p<0.01 and # = p<0.05.

Figure 3. Reactive oxygen species (ROS) production by BAL and bone marrow cells as well as splenic macrophages

BAL and bone marrow cells as well as splenic macrophages were harvested and enriched from naïve, sham-treated and CLP mice three (Panels A, C, F) and seven days (Panels B, D, E) after sepsis, and stimulated ex vivo with PMA in the presence of dihydrorhodamine at 37° C for up to 30 mins. Rhodamine fluorescence was gated on neutrophil enriched populations and presented as a percentage of control fluorescence. Data from CCR2^−/− mice are included in Panels E and F. Values represent the mean of 7-10 animals per group. Each experiment was confirmed three times. Significance between sham and CLP over time was determined by two way ANOVA* = p<0.01.

Figure 4. Outcome and colonization of p47^−/− mice infected with Pseudomonas pneumonia

(Panel A) Naïve C57BL/6 and p47^−/− mice were subjected to intranasal instillation of Pseudomonas and 24 hours later bacterial colonization of their BAL fluid was determined. Pseudomonas colonies in the naïve p47^−/− mice were comparable to levels seen in mice three days after sepsis and markedly higher than levels seen in sham-treated or control wild-type mice despite normal numbers of BAL neutrophils (Panels A, B and C). All p47^−/− mice succumbed to CLP prior to any intranasal instillation while healthy mice uniformly died in response to Pseudomonas (Panel D). Adoptive transfer of 5 × 10⁶ healthy naïve bone marrow neutrophils into p47^−/− mice reduced Pseudomonas colonization of the lungs (Panel A). Survival studies were carried out with 20 mice per group while 7 mice group were used for BALF bacterial and neutrophil determination. Significance in survival was determined by Fisher’s exact test and in BAL cfus and neutrophil counts by one way ANOVA. * = p<0.01

Figure 5. Liver and spleen colonization and outcome following listeriosis in septic, sham and control mice

Three and seven days (Panels A-D) after CLP-induced sepsis, mice were challenged with a sublethal L. monocytogenes infection (10⁴ cfu). Five days later, L. monocytogenes colony counts were determined in the spleen and liver. Animals that had sepsis induced three and seven days earlier had markedly reduced L. monocytogenes infection in the spleen and liver. When sham and septic mice were challenged with a lethal number of L. monocytogenes (10⁶ cfu), only the septic animals survived (Panels E, F). Bacterial colony counts represent the mean of seven animals per group, and the survival studies contained n=20 mice per group. Significance between sham and CLP liver and spleen cfu was determined by one way ANOVA, while survival differences were determined by Fisher’s exact test, * = p<0.05

Figure 6. Survival to L. monocytogenes infection in RAG^−/−, CCR2^−/− and MCP-2^−/− mice after polymicrobial sepsis

Wild-type and RAG^−/−, CCR2^−/− and MCP-2^−/− mice underwent CLP to induce sepsis and three days later, septic, sham-treated and control mice were administered an otherwise lethal L. monocytogenes infection (10⁶ cfu) (Panels A and B). Both wild-type and RAG^−/− mice with sepsis survived the lethal L. monocytogenes challenge (Panel A). Conversely, neither CCR2^−/− and MCP-2^−/− mice were protected from the lethality of L. monocytogenes infection by prior sepsis (Panel B). Values represent 20 animals per group. Each survival experiment was conducted 4 independent times. Significance between wild-type, RAG^−/−, CCR2^−/− and MCP-2^−/− mice was determined by Fisher’s exact test.

Figure 7. Blood monocyte and tissue macrophage response to polymicrobial sepsis

At intervals after polymicrobial sepsis, mice were sacrificed and blood and bone marrow CD11b⁺Ly6C⁺ cells were determined by flow cytometry Panels A presents total CD11b⁺Ly6C⁺ numbers in blood while Panels B-C represent bone marrow CD11b⁺Ly6C⁺F4/80⁺ percentages and absolute numbers. Values represent the mean of five to seven animals per group. Each experiment was confirmed at least three times. Significance between naïve, sham and CLP at day 3 and 7 was determined by one way ANOVA. Significance between sham and CLP over time was determined by two way ANOVA. * = p<0.01 and # = p<0.05.