Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.

Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5 × 10(5) CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.

Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.

Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae–induced pneumonia in mice. Rip2^−/− mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-γ levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2^−/− mice compared to wild-type (WT) mice at day 3. Rip2^−/− mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1^−/− and Nod2^−/− mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2^−/− mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.Author SummaryChlamydophila pneumoniae (C. pneumoniae) is a common intracellular parasite that causes lung infections and contributes to several diseases characterized by chronic inflammation. Toll-like receptors expressed on the cell surface detect C. pneumoniae and mount a vigorous defense, but it is not known how the cell defends itself once the pathogen has taken up residence as a parasite. We reasoned that cytosolic pattern recognition receptors called Nods (nucleotide oligomerization domain) that detect microbes that gain entry into the cell might be involved. Using mice genetically deficient in Nod1 and Nod2 or their common downstream adaptor (Rip2), we show that in lung infection, Nod proteins are indeed essential in directing a defense against C. pneumoniae. Mice with defective Nod/Rip2-dependent signaling exhibited delayed recruitment of neutrophils, blunted production of pro-inflammatory cytokines and chemokines, and evidence of defective iNOS expression and NO production. These impaired responses led to delayed clearance of bacteria, intense persistent lung inflammation, and increased mortality. By performing bone marrow transplantation experiments and direct transfer of cells into the lungs of mice, we demonstrated that intact Nod-dependent signaling in bone marrow–derived cells was critical in the defense against C. pneumoniae. Our results indicate that Nod proteins also play an important role in host defense against C. pneumoniae. Coordinated and sequential activation of TLR and Nod signaling pathways may be necessary for an efficient immune response and host defense against C. pneumoniae.