Pulmonary hypertension can be a sequela of prior Pneumocystis pneumonia

Abstract

Improved treatment regimens have reduced fatalities from opportunistic diseases, such as Pneumocystis pneumonia, in AIDS patients. However, serious chronic conditions, including pulmonary hypertension (PH), are increasing in this group. We report here that when CD4 T cells in Pneumocystis-infected mice are temporally depleted and then allowed to return, the extended inflammation results in PH that persists after Pneumocystis is eliminated. Using this model of PH, we have found that i) the onset of PH is correlated with the return of CD4 T cells, but PH persists after CD4 levels diminish; ii) vascular remodeling accompanies PH, but whereas temporary medial hypertrophy is evident with transient PH in immunocompetent mice, persistent PH is associated with perivascular fibrosis; iii) elevated levels of the fibrotic mediator FIZZ1 are found in bronchoalveolar lavage fluid of mice with persistent PH; and iv) although Th2-related mechanisms may be involved in PH etiology, PH still occurs in interleukin-4 receptor-deficient mice under these conditions. Overall, the data presented here demonstrate that the immune response to an infectious disease pathogen, such as Pneumocystis, can, when perturbed and prolonged, lead to later development of a serious chronic condition such as PH.

Figure 1

PH and perivascular inflammation is transient in immunocompetent mice. A: Pulmonary tissue in an immunocompetent mouse 14 days after inoculation with Pneumocystis showing marked perivascular inflammation (arrowhead) and enlarged medial layers of small pulmonary arteries (arrow). B: Similar view at 30 days after infection with limited residual perivascular inflammation and reduced medial hypertrophy in small arteries. C: Comparable view in an uninfected control. D and E: At 14 days after inoculation with Pneumocystis, mean peak right ventricular pressure (RVP) is significantly elevated, whereas the mass of the RV, expressed as the percentage RV mass is of the combined mass of the left ventricle + septum (LV + S), is slightly, but not significantly higher; n = 9 (infected) or 6 (control). F: At 30 days after infection, RVP is no longer significantly elevated [n = 6 (infected) or 7 (control)]. NS, nonsignificant. G: Inflammatory cells in the BALF are greatly reduced at 30 days compared with 14 days (Macs, macrophages; CD4s and CD8s are lymphocytes only; PMNs, neutrophils; EOS, eosinophils). Values are means ± SEM; P is probability that value is statistically equal to control animals. Original magnifications, ×400.

Figure 2

Mean peak RVP at 28 to 38 days after Pneumocystis inoculation with short-term CD4 depletion and Pneumocystis infection were evaluated (20 to 30 days after last anti-CD4 treatment). Recovery of CD4 T cells is highly variable, as was measured RVP. RVP is not correlated to Pneumocystis, but a correlation exists between RVP and CD4 levels. Points represent individual animals (n = 16) pooled from three separate experiments.

Figure 3

PH persists in BALB/c mice after Pneumocystis infection is cleared, when CD4 T cells have been temporally depleted. Pneumocystis-infected mice were depleted of CD4 cells for the first 10 days of infection; CD4 cells were then allowed to return. Both mean maximum RVP (A) and RV mass (B) remain elevated at 51 and 63 days after infection, even as the numbers of inflammatory cells decrease during this period (C). CD4⁺ and CD8⁺ are single positive lymphocyte populations, whereas DP lymphocytes are double positive for both CD4 and CD8. Values are means ± SEM, n = 12 (PC-infected 51 days and control) or n = 9 (infected 63 days); P is probability that value is statistically equal to control, noninfected mice.

Figure 4

Medial hypertrophy and inflammation in BALB/c mice subsides after Pneumocystis infection has been cleared. In mice with short-term CD4 depletion, perivascular inflammation (arrowhead) and thickness of the medial smooth muscle layer (arrows) are reduced from what was seen in 14-day infected immunocompetent mice (Figure 1A) at 51 (A) and 63 (B) days after infection, although both of these still have slightly greater area than in immunocompetent mice 45 days after infection (C) or control noninfected animals (D). Quantification of the smooth muscle layer confirms these observations (E and F); values represent the percentage of the diameter of the vessels that is medial smooth muscle (see Materials and Methods for details on calculations). E: Vessels larger than 50 μm but smaller than 100 μm in o.d. F: Vessels smaller than 50 μm. Values are means ± SEM, n = 12 (51 days infected and control) or 9 (63 and 14 days infected); P is probability that value is statistically equal to control. NS, nonsignificant.

Figure 5

Perivascular fibrosis in BALB/c mice after Pneumocystis infection has been cleared. Masson trichrome stains of sections of pulmonary tissue of Pneumocystis-infected (A and C) and control (B and D) BALB/c mice. Infected mice were CD4 depleted for the first 10 days of infection; samples were taken 63 days after inoculation. Increased staining density of extracellular matrix proteins is evident both in the cross section (A) and tangential section (C) of the infected lung (arrowheads, collagen stains blue with this procedure). Quantification of soluble collagen levels in short-term CD4-depleted mice at 51 and 62 days after inoculation confirms the higher levels of extracellular matrix protein in lung tissue. E: Lung homogenates were assayed for soluble collagen as described in Materials and Methods. Results are expressed as micrograms of soluble collagen per milligram of total protein in the sample, means ± SEM, n = 7 per group. Increases in BALF levels of the fibrotic mediator FIZZ1 coincided with increased fibrosis (F); aliquots of BALF were tested by ELISA for the presence of the putative fibrotic mediator and quantified against standards of FIZZ1 peptide. Values are means ± SEM, n = 8 to 11. Original magnifications, ×400.

Figure 6

Persistent PH does not depend on IL-4 signaling. IL-4 KO mice were Pneumocystis-infected and depleted of CD4 cells for 10 days and then sampled at 57 days after inoculation. A: RVP of infected and control IL-4 KO mice. B: Relative right ventricular mass of infected and control IL-4 KO mice. These events occur despite different cellular response to Pneumocystis, as indicated by the profile of BAL cells seen 14 days after infection (C); when compared with immunocompetent mice (see Figure 1G), there is no eosinophil response but a higher recruitment of neutrophils. Values are means ± SEM, n = 5; P is probability that value is statistically equal to control.