Lung infections cause prolonged immune alterations and elevated susceptibility to secondary pneumonia. We found that, after resolution of primary viral or bacterial pneumonia, dendritic cells (DC), and macrophages exhibited poor antigen-presentation capacity and secretion of immunogenic cytokines. Development of these “paralyzed” DCs and macrophages depended on the immunosuppressive microenvironment established upon resolution of primary infection, which involved regulatory T (Treg) cells and the cytokine TGF-β. Paralyzed DCs secreted TGF-β and induced local Treg cell accumulation. They also expressed lower amounts of IRF4, a transcription factor associated with increased antigen-presentation capacity, and higher amounts of Blimp1, a transcription factor associated with tolerogenic functions, than DCs present during primary infection. Blimp1 expression in DC of humans suffering sepsis or trauma correlated with severity and complicated outcomes. Our findings describe mechanisms underlying sepsis- and trauma-induced immunosuppression, reveal prognostic markers of susceptibility to secondary infections and identify potential targets for therapeutic intervention.

As macrophages and dendritic cells (DC) orchestrate immunity and tolerance, we compared their functional properties before, during, and after resolution of pneumonia. Both cell types showed profound alterations—which we summarize as “paralysis”—in the latter case. Paralysis was caused by excessive release of local mediators of restoration of immune homeostasis. Our findings suggest that DC and macrophage dysfunction is an important contributor to protracted immunosuppression after bacterial or viral primary sepsis and increased susceptibility to secondary pneumonia.

Healthy lungs are colonized by bacteria whose burden is continuously controlled by mucosal immunity (). Infection by pathogenic bacteria disrupts this balance and can induce lung injury through direct damage caused by the pathogen, or through immunopathology elicited by the effector mechanisms of immunity. Therefore, a healthy immune response should maximize the deployment of effector mechanisms against the pathogen while minimizing the damage of self-tissues that might ensue.

Pneumonia is the leading cause of death from infectious disease (). The risk of developing pneumonia increases following severe primary infections and reaches 30%–50% for critically ill patients recovering from a first episode of infection (). It is currently accepted that susceptibility to secondary pneumonia increases due to acquired immune defects collectively known as sepsis-induced immunosuppression (). In-depth understanding of the mechanisms involved is vital to prevent and treat secondary pneumonia in patients recovering from a primary infection.

Since we found in mice that the reprogramming of DC is not pathogen-driven but induced by secondary mediators of inflammation, we reasoned that Blimp1CD1c DC might also be observed in patients suffering aseptic SIRS, such as severe trauma patients, who are highly susceptible to secondary pneumonia (). We thus examined circulating DC from patients suffering from trauma-induced severe inflammation (IBIS cohorts 1 and 2, n-32 and n-29 respectively Table S2 ). We have previously shown that circulating DC from these patients have lost their ability to produce TNF-α, IL-6, and IL-12 upon in vitro stimulation (), reproducing a hallmark of mouse paralyzed DC ( Figures 4 E and 4F). Blimp1 expression was also increased in circulating CD1c DC collected from these trauma patients as compared to matched healthy controls ( Figure 6 B). The level of expression of Blimp1 in circulating CD1c DC increased with the severity of the trauma ( Figure 6 C) and correlated with the duration of mechanical ventilation, a surrogate marker of complicated outcome ( Figure 6 D). We also found an increase in the number and frequency of circulating Treg cells in trauma patients ( Figure 6 E), which again correlated with trauma severity ( Figure 6 F).

Finally, we addressed whether the conclusions of our studies in mouse models of infection could be extrapolated to the human system. First, we analyzed PBMC from a prospective cohort of septic patients presenting with E. coli secondary infection (IBIS sepsis, n = 5 [ Table S1 ]). Human CD1c DC, the circulating equivalent of murine CD11b DC (), expressed high level of the transcription factor Blimp1 in these patients as compared to matched uninfected donors ( Figure 6 A), paralleling the result observed in mice. Also reproducing what we observed in infected-cured mice ( Figure 4 H), CD141 DC (the human equivalent of murine CD103DC) did not show increased Blimp1 expression in these patients ( Figure 6 A).

(F) Correlation between the accumulation of Treg (Delta = number at day 7 – number at day 1) and trauma severity in brain-injured patients. Glasgow Coma Scale rates the severity of the brain-injury from 15 (minor injury) down to 3 (major injury).

(E) Number and frequency of circulating CD4 Treg in brain-injured patients suffering from trauma-induced systemic inflammation. Blood samples were collected 1 and 7 days after the trauma (n = 27 severe trauma patients).

(C and D) Correlation between Blimp1 expression in circulating CD1c DC and (C) trauma severity (Glasgow Coma Scale) or (D) duration of mechanical ventilation (days) in brain-injured patients with trauma-induced systemic inflammation. Glasgow Coma Scale rates the severity of the brain-injury from 15 (minor injury) down to 3 (major injury).

(B) Expression of Blimp1 in circulating CD1c DC collected in healthy controls and in brain-injured patients with trauma-induced systemic inflammation. Blood samples were collected 7 days after the trauma (n = 15 controls and n = 32 severe trauma patients).

(A) Expression of Blimp1 in circulating CD1c DC and CD141 DC of uninfected donors and of patients presenting severe secondary infection (n = 12 controls and n = 5 patients with severe secondary infections).

Blimp1 Expression in CD1c DC and Treg Accumulation Are Correlated with the Disease Severity of Humans Presenting with Systemic Inflammatory Response

Figure 6 Blimp1 Expression in CD1c DC and Treg Accumulation Are Correlated with the Disease Severity of Humans Presenting with Systemic Inflammatory Response

Finally, we examined whether reduced cytokine production by DC and macrophages during secondary pneumonia was also caused by TGF-β recognition by the cells themselves. This did not appear to be the case because in WT:Tgfbr2Cd11cmixed bone marrow chimeras, both the WT and the TGF-βR-deficient cells were impaired during secondary pneumonia ( Figure 5 F). Because neutralization of TGF-β rescued IL-12 and IL-6 production by DC and macrophages ( Figures 5 B and 5C), this suggested that TGF-β acted through an indirect mechanism to impair cytokine production by the two cell types. Treg cells are known to inhibit DC functions () and depletion of Treg cells during the resolution of primary pneumonia enhanced IL-12 and IL-6 production by macrophages and DC during secondary infection ( Figure 5 G). Together, our results indicate a pivotal role for TGF-β in the induction of DC and macrophages with reduced immunogenic function. It acts directly on developing cells, and indirectly via Treg cells.

In order to investigate the role of TGF-β signaling in macrophages and DC on their ability to prime CD4 T cells, we generated mixed bone marrow chimeras where WT mice were reconstituted with a 1:3 mix of Tgfbr2Cd11cand H2(MHC-II-deficient) bone marrow. In these chimeric mice, only the cells derived from the Tgfbr2Cd11cbone marrow are able to present antigen to, and prime, CD4 T cells. TGF-βR-deficient CD11c cells retained the ability to elicit effective priming of OT-II cells during secondary pneumonia in vivo ( Figure 5 E).

We reasoned that since TGF-β influences acquisition of functional properties during DC development (), its production in mice recovered from primary infection might contribute to the local imprinting of DC toward tolerance. Neutralization of TGF-β with a blocking mAb injected during, or after resolution of, primary pneumonia reduced the defects in DC cytokine production during secondary infection ( Figures 5 B and 5C). To explore further the role of TGF-β signaling on DC modulation, we used Tgfbr2Cd11cmice, which lack expression of TGF-β receptor selectively in DC and macrophages. These mice spontaneously succumb to inflammatory disease (), so we generated mixed bone marrow chimeras where WT mice were reconstituted with a 1:3 mix of Tgfbr2Cd11cand WT bone marrow. Seven days after E. coli infection, the proportion of TGF-βR-deficient CD11c cells (CD45.2cells) in the lungs of these mice was significantly lower than in uninfected chimeras ( Figure 5 D), confirming the role of TGF-β in macrophage and DC renewal after infection.

To examine whether the signals responsible for reprogramming of DC after pneumonia acted systemically, we assessed the phenotype and function of splenic DC 7 days following primary pneumonia. Neither the expression of transcription factors ( Figure 4 I), nor capacity for T cell priming and cytokine production of these cells were altered 7 days after E. coli pneumonia ( Figures 4 J and 4K). Therefore, signals that induce altered DC functions after recovery from primary pneumonia only act locally on cells that undergo terminal differentiation in the same tissues that suffered the infection. Next we addressed whether such signals consisted of pathogen-derived products that lingered at the infection site (), or of endogenous mediators produced by the affected tissues (). We generated mixed-bone marrow chimeras where wild-type (WT) mice received a 1:1 ratio of bone marrow from WT (CD45.1) or Tlr9(CD45.1) donors ( Figure S7 A). In this setting, Tlr9cells cannot recognize the pathogen-associated molecular pattern mimic CpG, but can receive signals from secondary mediators released by WT cells responding to CpG (). Intra-tracheal administration of CpG induced a lung inflammatory response that caused lung DC and macrophage activation, followed by a 7-day long recovery phase in which the activated cells were replaced by immature cells ( Figures S7 B and S7C), reproducing the time course of the recovery from E. coli or IAV infection. At day 7 post-CpG treatment, we challenged the chimeric animals with E. coli and measured cytokine production by WT and Tlr9DC or macrophages, finding both groups of cells displayed reduced production of IL-12 and IL-6 compared to their counterparts from naive mice ( Figure 5 A). This result implied that the functional alterations observed in DC and macrophages following recovery from pneumonia were induced not by direct encounter of pathogen products but by secondary mediators of inflammation.

p < 0.05,p < 0.001, #p < 0.05 versus all others. Graphs represent mean ± SD and display data pooled from 2–3 independent experiments. See also Figures S7

(G) Frequency of IL12 + alveolar macrophages, IL12 + CD103 DC and of IL6 + CD11b DC during primary (1ary) or secondary (2ary) pneumonia in wild-type (WT) or DT-treated DEREG mice (DT 0.1 mg i.p. day+4 and day+6 after 1ary pneumonia) (n > 6 mice per group).

(F) WT (CD45.1 + ): Tgfbr2 fl/fl Cd11c cre (CD45.2 + ) chimeras E. coli infection-cured were re-challenged with E. coli (2ary pneumonia). Frequency of IL12 + alveolar macrophages, IL12 + CD103 DC and of IL6 + CD11b DC was determined in WT and TGF-βRII-deficient cells (n = 4 mice per group).

(E) Lethally irradiated WT recipient mice were reconstituted with 3:1 H2 −/− (which produces MHC-II deficient cells unable to induce CD4 T cell proliferation) and CD45.2 + Tgfbr2 fl/fl Cd11c cre bone marrow. Eight weeks after immune reconstitution, Cell Trace Violet labeled OT-II (i.v.) and OVA-coated E. coli (intra-tracheal) were injected in naive (primary, 1ary pneumonia) or E-coli infection-cured (secondary, 2ary pneumonia) chimeras. 60 hr later, the proliferation of OT-II was assessed in the mediastinal lymph nodes (n = 5–6 mice per group).

(D) Lethally irradiated WT recipient mice were reconstituted with a 3:1 ration of CD45.1 + WT and CD45.2 + Tgfbr2 fl/fl Cd11c cre (which produces TGF-βRII-deficient DC). Eight weeks after immune reconstitution, the percentage of CD45.2 + TGF-βRII-deficient macrophages and DC was assessed in the lungs of uninfected of E. coli infection-cured chimeras (n = 4 mice per group).

(B and C) Percentage of IL12 + CD103 DC and IL6 + CD11b DC during secondary (2ary) pneumonia elicited 7 days after primary (1ary) pneumonia in WT mice treated with anti-TGF-β or isotype control monoclonal antibody (44 μg i.p.) (B) during (day+3 and day+6) or after (day+7 and day+10) resolution of 1ary pneumonia (n = 3–6 mice per group).

(A) Frequency of IL12 + CD103 DC, IL12 + alveolar macrophages and IL6 + CD11b DC was measured after induction of E. coli pneumonia in WT+Tlr9 −/− mixed bone marrow chimeras (1:1 ratio) intratracheally injected (so-called secondary pneumonia, 2ary PN) or not (so-called primary pneumonia, 1ary PN) with CpG 7 days prior (n = 4 mice per group).

Next we compared the expression of phenotypic markers and immunoregulatory factors in DC before and after pneumonia. We found no significant changes in the expression of characteristic surface markers of DC (CD11c, CD24, MHC-II, DEC205, CD103, and CD11b) and macrophages (F4/80, CD64, Ly6G, CD11c, CD11b) ( Figures S6 A and S6B). In contrast, the amounts of key transcription factors involved in the control of immunogenic versus tolerogenic functions of DCs were significantly altered () ( Figure 4 H). Specifically, the amount of IRF4, which promotes antigen presentation to CD4 T cells (), was lower in CD11bDC and in CD103DC after clearance of the infection ( Figure 4 H). Conversely, expression of the transcription factor Blimp1, which induces tolerogenic functions in DC (), was increased ( Figure 4 H). Expression of two other transcription factors involved in DC development and whose expression in DC is critical for immune response to pathogens (), ID2 and IRF8, remained unchanged ( Figure 4 H). Expression of these four transcription factors did not change in macrophages ( Figure S6 C). Thus, although neither the rate of DC turn over ( Figure 4 B) nor the expression of characteristic surface markers of DC subtypes changed substantially after resolution of primary pneumonia, the expression of transcription factors that regulate DC function did.

Production of immunogenic cytokines by macrophages and DC is as, if not more, critical for the control of infection than antigen presentation, as these cytokines regulate both innate and T cell-dependent immunity (). We identified CD103DC, alveolar macrophages, and CD11bDC as the main sources of interleukin (IL)-12, tumor necrosis factor (TNF)-α and IL-6, respectively, during E. coli primary pneumonia ( Figure S5 A). Production of these cytokines during secondary pulmonary infection with E. coli was significantly impaired for up to 30 days in mice recovered from primary E. coli or IAV pneumonia ( Figures 4 E and 4F). This defect was apparent even if the dose of E. coli causing secondary pneumonia was increased 3.3 times ( Figure S5 B), or if the bacterium causing the secondary pneumonia was different to the one that caused the primary one (e.g., Staphylococcus aureus or Pseudomonas aeruginosa, Figure S5 C). IL-12 is required to elicit interferon (IFN)-γ production by NK cells ( Figure S5 D), a cytokine that in turn plays a critical role in the resolution of bacteria-induced pneumonia (). Treatment of mice with IL-12 during secondary pneumonia enhanced bacterial clearance ( Figure 4 G), and restored IFN-γ production by NK cells ( Figure S5 E). These results show that defective immunogenic cytokine production by macrophages and DC plays a central role in the increased susceptibility of infected-cured mice to secondary pneumonia.

Capture and presentation of pathogen antigens via MHC-II is a hallmark property of DC and macrophages (), and though their numbers during primary and secondary pneumonia were comparable ( Figure S4 A), MHC II-mediated T cell priming was defective in mice suffering secondary pneumonia for at least 21 days after recovery from primary infection ( Figures 1 E and 1F). Both macrophages and DC of mice that recovered from primary pneumonia showed defective antigen-presentation capacity in vitro ( Figure 4 A). We have shown that primary pneumonia causes systemic activation of lung DCs ( Figure S4 B), and since mature DCs cannot present newly encountered antigens (), persistent DC maturation might explain the lack of antigen presentation by DC in mice that recovered from primary pneumonia. However, neither DCs nor macrophages exhibited signs of activation (high CD86 expression) at that stage ( Figure S4 B). Moreover, measurements of BrDU incorporation showed the rate of macrophages and DC renewal in mice recovered from primary pneumonia was comparable to that in non-infected mice ( Figure 4 B). This suggested that, as the mice recovered from primary pneumonia, activated DC and macrophages were replaced by “immature” cells that were defective at detecting and/or presenting antigen from a secondarily infecting pathogen. Both DC and macrophages increased CD86 expression at the onset of secondary infection with E. coli in the lungs ( Figures S4 C and S4D), demonstrating they were still responsive to pathogens. Targeting antigen to a surface DC receptor can overcome defects in antigen presentation (), and we observed effective OT-II priming in mice recovered from primary pneumonia if they received OVA conjugated to a mAb that recognizes the DC receptor, DEC-205 ( Figure 4 C). Furthermore, OT-II priming occurred in infection-cured CD11c-OVA transgenic mice challenged with a secondary E. coli infection, in which macrophages and DC constitutively express and present OVA ( Figure 4 D). Therefore, OT-II activation and induction of proliferation can occur in mice suffering a secondary infection if the T cells encounter their cognate MHC-peptide complex on the surface of CD11ccells (DC). This series of experiments demonstrate that DC and macrophages, which continually turn over in the lungs (), develop with impaired capacity to capture, process, and/or generate MHC-II-peptide complexes for 21 days or more after recovery from primary pneumonia.

p < 0.05,p < 0.01,p < 0.001, #p < 0.01 versus all other groups, One-way ANOVA. Graphs represent mean ± SD and are pooled data from 2–3 independent experiments. See also Figures S4–S6

(J and K) E. coli infection-cured mice were intravenously injected with (J) soluble OVA plus Cell Trace Violet labeled OT-II and OT-II proliferation in the spleen was assessed 60 hr later; or (K) with CpG (20 nM i.v.) or LPS (1 μg i.v.) and frequency of splenic IL12 + CD8 DC was measured 2 hr later.

(I) Expression of IRF-4 in splenic DC of WT mice, and of ID2, Blimp1 and IRF8 in specific reporter mice left uninfected or infected 7 days previously with E. coli (infection-cured). (n = 3–4 per group).

(H) Expression of IRF-4 in lung DC of WT mice, and of ID2, Blimp1 and IRF8 in specific reporter mice (ID2 GFP , Blimp1 GFP and IRF-8 YFP respectively) left uninfected or infected 7 days previously with E. coli (infection-cured). (n = 6 for IRF-4, and = 3–4 for reporter mice).

(G) Enumeration of CFU from bronchoalveolar lavages 16 hr following intra-tracheal injection of E. coli (75 μL of DH5α intra-tracheal, OD 600 = 0.6) in either naive mice (primary, 1ary pneumonia), or infection-cured (2ary pneumonia) with or without IL-12 treatment (100 ng i.p.) concurrent with induction of 2ary pneumonia (n = 4–6 independent mice).

(E and F) Frequencies of IL-12 + CD103 DC, TNF-α + alveolar macrophages and IL6 + CD11b DC during primary (1ary) E. coli pneumonia (75 μL of DH5α intra-tracheal, OD 600 = 0.6) or secondary (2ary) E. coli pneumonia realized at the indicated time point after (E) E. coli or (F) Influenza A Virus (IAV) 1ary pneumonia (n > 8 mice at day 7, n = 2–3 at day 14, 21, 30, and 45).

(D) Cell Trace Violet-labeled OT-II cells (i.v.) and E. coli (intra-tracheal) were administered to CD11c-OVA mice that were naive (1ary pneumonia) or E. coli infection-cured (2ary pneumonia). OT-II proliferation was assessed 60 hr later in the mediastinal lymph node (n = 5 mice per group).

(C) Cell Trace Violet-labeled OT-II cells (iv.) and anti-DEC205-OVA (intra-tracheal) were delivered to WT mice that were naive (uninfected) or infected 7 days prior with E. coli (infection-cured). OT-II proliferation was assessed 60 hr later in the mediastinal lymph node (n = 5 mice per group).

(B) BrDU was injected i.p. (1 mg per day for 2 days) in uninfected WT mice, or in mice infected with E. coli 5–7 days earlier (infection-cured). Percentage of BrDU + lung macrophages and DC was assessed by flow cytometry (n = 3 mice per group).

(A) Number of OT-II cells after 60 hr of in vitro co-culture of naive OT-II (50 × 10 3 cells) with increasing doses of soluble OVA and either (a) macrophages or (b) DC (10 × 10 3 cells) collected from lungs of naive mice or mice infected with E. coli 7 days prior (infection-cured) (n = 2 independent experiments, data is pooled with 5 mice per group for macrophages and DC donors).

DC and macrophages become activated, increase in numbers ( Figures S3 D and S3E) and elicit protective immunity ( Figures S3 F and S3G) against primary E. coli infection, yet as shown above these cells appear critical in the induction of tolerance to secondary infection. We therefore sought to compare further the function and phenotype of these two cell types before, during, and after primary pneumonia.

Next we sought to identify the cells that produced TGF-β in the lungs of mice cured from primary infection. Expression of TGF-β mRNA did not vary in non-hematopoietic cells (CD45) in infection-cured mice ( Figure S2 H), suggesting the cells responsible were hematopoietic. Macrophages and DC produce and activate TGF-β, inducing Treg cell formation (), and the Treg cells that accumulated in infection-cured mice were neuropilin Figure S2 I), indicating they were peripherally induced rather than thymus-derived, natural Treg (). Indeed, we observed increased TGF-β mRNA expression in lung DC of mice recovered from primary pneumonia ( Figure 3 A), although expression of RNA for TGF-β activators remained unchanged ( Figure S2 J). Production of TGF-β protein, as assessed by the membrane expression of its inactive precursor Latency-Associated Peptide (LAP) (), was increased in CD11bDC of mice cured from primary pneumonia compared to DC of naive mice ( Figure 3 B). This correlated with the ability of these DC to induce Treg cells in vitro ( Figure 3 C). We used CD11c-DTR transgenic mice, where we could deplete both macrophages and DC ( Figures S3 A and S3B), to test their role in Treg cell induction. Their depletion did not affect the number of lung CD4 T cells ( Figure S3 C), but it reduced the number of Treg cells in mice recovered from primary pneumonia ( Figure 3 D) or suffering secondary pneumonia ( Figure 3 E). This reduction was reversed with anti-TGF-β treatment ( Figure 3 D, red dots). Altogether, these results indicate that DC and macrophages of mice that recovered from primary pneumonia produce TGF-β and promote Treg cell differentiation.

p < 0.05,p < 0.01. Graphs represent mean ± SD and are a compilation of 2–3 independent experiments. See also Figures S2 H–S2J and S3

(E) Frequency and number of lung FoxP3 + CD4 T cells during secondary (2ary) E. coli pneumonia realized in CD11c-DTR mice treated or not with DT after cure from E. coli infection (0.1 μg i.p. day+6 and +7 after 1ary E. coli pneumonia) (each dot represent an independent biological replicate, n = 5–6 mice per group).

(D) Frequencies and number of lung FoxP3 + CD4 T cells in CD11c-DTR chimeric mice cured from E. coli infection, treated or not with DT (0.1 μg ip. day-1, day 0 then every 3 days) and injected or not with TGF-β treatment (1 μg i.p at day +6) (n = 6–8 mice per group, except n = 4 for TGF-β treatment).

(C) Frequency and number of CD4 + FoxP3 + Treg cells after 5 days of in vitro co-culture of naive OT-II cells (50 × 10 3 cells) with soluble OVA and either (I) macrophages or (II) DC (10 × 10 3 cells) isolated from lungs of naive mice or E. coli infection-cured mice (n = 2 independent experiments with 5 pooled mice per biological replicate).

(B) Membrane expression of Latency Associated Peptide (LAP), inactive form TGF-β, by lung macrophages and DC from WT mice infected or not with E. coli or IAV 7 days prior and considered as infection-cured (n = 4–6 mice per group).

(A) Relative expression of TGF-β mRNA in macrophages and DC purified by flow cytometry from lungs of WT mice infected or not with E. coli 7 days prior (infection-cured) (n = 3 independent biological replicates per group from 4–5 pooled mice).

TGF-β induces differentiation of naive CD4 T cells into FoxP3T regulatory (Treg) cells (). We found a higher proportion of lung Treg cells after recovery from primary bacterial or IAV pneumonia ( Figures S2 A and S2B), and also in the lungs of mice suffering secondary pneumonia ( Figure 2 C), than in mice uninfected or suffering primary pneumonia. Treatment with anti-TGF-β reduced Treg cells accumulation ( Figure 2 D), so we investigated the role of Treg cells in susceptibility to secondary infection. We infected transgenic mice expressing the diphtheria toxin receptor (DTR) in FoxP3cells (DEREG mice), where we could deplete Treg cells after initiation of primary or secondary pneumonia ( Figure S2 C). Depletion of Treg cells during the resolution of primary pneumonia (from days 4 to 7 post primary infection) did not alter the course of this infection ( Figures S2 D and S2E), but restored the effectiveness of bacterial clearance, and enhanced CD4T cell priming, during secondary pneumonia ( Figures 2 E and 2F). Thus, TGF-β in the lungs of mice that recovered from primary pneumonia induced Treg cell accumulation during secondary pneumonia, which contributed to immunosuppression.

Tumor growth factor (TGF)-β is critical for tissue healing after injury and is immunosuppressive (). To test whether TGF-β released within lung tissue during or after primary pneumonia induced immunosuppression, we neutralized it with a mAb injected 3 and 6 days after initiation of primary pneumonia. This treatment did not affect bacterial burden or weight changes during primary infection ( Figures S1 A–S1D), but it caused reduced bacterial burden and increased OT-II cell priming during secondary pneumonia ( Figures 2 A and 2B ). This indicated a role for TGF-β on the induction of immunosuppression after recovery from primary infection.

p < 0.05,p < 0.01, # p < 0.05 versus all others. Graphs represent mean ± SD and are a compilation of 2–3 independent experiments. See also Figures S1 and S2 A–S2G.

(F) 7 days E. coli primary pneumonia, WT, or DT-treated DEREG mice (DT 0.1 mg ip. day+4 and day+6 after 1ary pneumonia) were subsequently injected with OVA-coated E. coli (intra-tracheal) and Cell Trace Violet labeled OT-II (secondary, 2ary pneumonia). OT-II proliferation was assessed 60 hr later in the mediastinal lymph node (n = 5–6 mice per group).

(E) Number of CFU per milliliter of bronchoalveolar lavage (BAL) during primary (1ary) or secondary (2ary) pneumonia in WT or DT-treated DEREG mice (DT 0.1 mg ip. day+4 and day+6 after 1ary pneumonia) (n = 3–4 mice per group).

(D) Number and frequency of lung FoxP3 + CD4 T cells in WT mice treated with anti-TGFβ or isotype control monoclonal antibody after primary pneumonia (44 μg ip. at day +3 and day +6), and subsequently challenged (day+7) with secondary pneumonia (n = 5–6 mice per group).

(C) Frequency and number of lung FoxP3 + CD4 T cells in WT mice that were uninfected, or infected with E. coli to elicit primary pneumonia (1ary PN, black dots), or following secondary E. coli pneumonia (2ary PN, gray dots) elicited 7 days following (I) E. coli or (II) IAV (each dot represents an independent biological replicate).

(B) WT mice were treated with anti-TGF-β or isotype control monoclonal antibody after primary (1ary) E. coli pneumonia (44 μg i.p. at day+3 and day+6), and subsequently injected (day+7) with OVA-coated E. coli (intra-tracheal) and Cell Trace Violet labeled OT-II (secondary, 2ary pneumonia). OT-II proliferation was assessed 60 hr later in the mediastinal lymph node (n = 5–6 mice per group).

(A) WT mice were treated with anti-TGF-β or isotype control monoclonal antibody after primary (1ary) E. coli pneumonia (44 μg i.p. at day+3 and day+6) then injected with E. coli at day+7 (2ary pneumonia). The number of CFU per milliliter of bronchoalveolar lavage (BAL) was assessed 18 hr later (n ≥ 5 mice per group).

We first assessed T cell priming in mice that recovered from bacterial pneumonia by re-infecting them 7–21 days after the primary infection with E. coli associated with the model antigen, ovalbumin (OVA). The mice also received MHC II-restricted, OVA-specific OT-II cells, which proliferated in the mediastinal lymph nodes (LN) in response to local presentation of OVA. We observed a severe reduction in OT-II proliferation during secondary pneumonia compared to that observed in mice that received E. coli-OVA as a primary infection ( Figure 1 F). Similar results were obtained in mice where primary pneumonia was caused by IAV ( Figure 1 G).

Escherichia coli (E. coli) is the second most frequent gram negative bacilli involved in both community- and hospital-acquired pneumonia (). Early recurrence of pneumonia to the same pathogens is observed in up to 20% of critically ill patients cured from primary pneumonia (). To mimic in mice this clinical scenario, we induced secondary pneumonia with E. coli in mice cured from a bacterial (E. coli) or a viral (influenza A virus, IAV) primary pneumonia ( Figure 1 A). During primary E. coli pneumonia, pathogen burden and mouse weight loss peaked 1 day after infection and then decreased until by day 7 the mice had cleared the bacterium ( Figure 1 B) and recovered their normal weight ( Figure 1 C). If these mice were re-infected with E. coli 7 to 21 days after the primary infection, they suffered more severe (secondary) pneumonia with increased bacterial burden and weight loss ( Figures 1 C and 1D). Similarly, mice infected with E. coli suffered more severe pneumonia if they had previously been infected with, and recovered from primary pneumonia caused by IAV (). We exploited this experimental model to characterize potential mechanisms of sepsis-induced immunosuppression ().

(F and G) At the indicated times after (F) E. coli or (G) IAV primary pneumonia, Cell Trace Violet-labeled OT-II cells (iv) and OVA-coated E. coli (intra-tracheal) were injected concomitantly in WT mice. OT-II proliferation was assessed 60 hr later in the mediastinal lymph node (n = 6–7 mice per group, except day 14 and day 21 n = 3).

(E) Enumeration of CFU per milliliter of bronchoalveolar lavage analyzed one day after secondary E. coli pneumonia (75 μL of DH5α intra-tracheal) induced 7 days after influenza A virus (IAV) primary pneumonia (n = 5 mice per group).

(D) Enumeration of colony-forming units (CFU) per milliliter of bronchoalveolar lavage (CFU/mL) analyzed one day after secondary E. coli pneumonia (75 μL of DH5α intra-tracheal, OD 600 = 0.6) induced at the indicated times after E. coli primary pneumonia (n = 6 mice per group).

(B) Time course of the bacterial load after intra-tracheal instillation of E. coli in naive mice (primary pneumonia) (n = 3 mice per group).

Recovery from Primary Pneumonia Is Followed by a Susceptibility to Secondary Pneumonia and Prolonged Reduction in Antigen-Presentation Function

Figure 1 Recovery from Primary Pneumonia Is Followed by a Susceptibility to Secondary Pneumonia and Prolonged Reduction in Antigen-Presentation Function

Discussion

The effector mechanisms deployed by the immune system to fight pathogens can cause tissue damage and have to be tightly controlled to prevent self-harm. Here we have described a network of regulatory mechanisms that dampen the immune response locally in response to lung infection. It involves multiple cell types and cytokines, with macrophages and DC playing a pivotal role. Importantly, after clearance of the infection the immunosuppression induced by these mechanisms does not restore immune homeostasis to the situation that preceded the infection. It persists locally for weeks after resolution of the infection, increasing the susceptibility to secondary infections.

Kim et al., 2011 Kim S.J.

Zou Y.R.

Goldstein J.

Reizis B.

Diamond B. Tolerogenic function of Blimp-1 in dendritic cells. high DC in critically ill patients might be a prognostic marker of extended immunosuppression, affording an opportunity for early intervention to prevent secondary infections in this high-risk cohort of patients. This might be achieved by reducing the exposure of the patients to potential pathogens or applying antibiotic prophylaxis ( Roquilly et al., 2015 Roquilly A.

Marret E.

Abraham E.

Asehnoune K. Pneumonia prevention to decrease mortality in intensive care unit: a systematic review and meta-analysis. We showed that circulating DC of sepsis or trauma patients express characteristic markers of mouse paralyzed DC such as a high level of Blimp1, a transcription factor associated with reduced immunogenicity (). The presence of Blimp1DC in critically ill patients might be a prognostic marker of extended immunosuppression, affording an opportunity for early intervention to prevent secondary infections in this high-risk cohort of patients. This might be achieved by reducing the exposure of the patients to potential pathogens or applying antibiotic prophylaxis (). Alternatively, treatments that reduce the formation of paralyzed DC such as blockade of TGF-β activity might reduce the duration of immunosuppression. Another point of intervention might be overcoming defects in cytokine production or antigen presentation; for example, treatment with immunogenic cytokines such as IL-12 had protective effects in mice and might exert similar benefits in humans.