Enhancing Cystic Fibrosis Immune Regulation

Abstract

In cystic fibrosis (CF), sustained infection and exuberant inflammation results in debilitating and often fatal lung disease. Advancement in CF therapeutics has provided successful treatment regimens for a variety of clinical consequences in CF; however effective means to treat the pulmonary infection and inflammation continues to be problematic. Even with the successful development of small molecule cystic fibrosis transmembrane conductance regulator (CFTR) correctors and potentiators, there is only a modest effect on established infection and inflammation in CF patients. In the pursuit of therapeutics to treat inflammation, the conundrum to address is how to overcome the inflammatory response without jeopardizing the required immunity to manage pathogens and prevent infection. The key therapeutic would have the capacity to dull the inflammatory response, while sustaining the ability to manage infections. Advances in cell-based therapy have opened up the avenue for dynamic and versatile immune interventions that may support this requirement. Cell based therapy has the capacity to augment the patient's own ability to manage their inflammatory status while at the same time sustaining anti-pathogen immunity. The studies highlighted in this manuscript outline the potential use of cell-based therapy for CF. The data demonstrate that 1) total bone marrow aspirates containing Cftr sufficient hematopoietic and mesenchymal stem cells (hMSCs) provide Cftr deficient mice >50% improvement in survival and improved management of infection and inflammation; 2) myeloid cells can provide sufficient Cftr to provide pre-clinical anti-inflammatory and antimicrobial benefit; 3) hMSCs provide significant improvement in survival and management of infection and inflammation in CF; 4) the combined interaction between macrophages and hMSCs can potentially enhance anti-inflammatory and antimicrobial support through manipulating PPARγ. These data support the development of optimized cell-based therapeutics to enhance CF patient's own immune repertoire and capacity to maintain the balance between inflammation and pathogen management.

Keywords: bone marrow transplantation; cystic fibrosis; hematopoietic cells; immune support; infection; inflammation; macrophages; mesenchymal stem cells.

FIGURE 1

Bone marrow non-congenic chimeras and response to chronic P. aeruginosa infection. CF→CF (closed triangles), WT→CF (open triangles; closed bars), CF→WT (closed circles; clear bars) and WT→WT (open circles) mice were inoculated with P. aeruginosa-laden agarose beads on Day 0, three months following bone marrow reconstitution. Mice were monitored daily following infection. Moribund mice were euthanized for humane reasons and included as if spontaneous death had occurred. (A) Significantly different survival rates on Day 10 between the four possible groups (p = 0.03; Fisher’s exact test) where donors and recipients were males. Significantly different survival rates on Day 10 with improvement in survival of CF animals with WT BM and decrease survival of WT mice with CF BM compared to survival of CF animals and WT animals without BM four possible groups (p = 0.0284; Fisher’s exact test). (B) BAL fluid was collected from one subset of WT→CF (N = 11), CF→WT (N = 10) mice sacrificed three days after P. aeruginosa challenge for cytokine and chemokine analysis. *Significantly different from CF → WT mice (p ≤ 0.03; Wilcoxon test). (C) Following BAL fluid collection in mice represented in Figure 1C, the areas of inflammation in the left and right lungs were evaluated using point counting. *Significantly different from CF → WT mice (p ≤ 0.01; Wilcoxon test). (D) The area of the location of the inflammation in the right lung is shown. *Significantly different from CF → WT mice (p = 0.03; Wilcoxon test). (E) The severity of the inflammation within the endobronchial spaces of the right lung is shown. *Significantly different from CF → WT mice (p = 0.04; Wilcoxon test). Data in the bar graphs are shown as the means ± SEM.

FIGURE 2

Bone marrow congenic chimeras and response to chronic P. aeruginosa infection. Bone marrow chimeras were generated using congenic CF (Cftr ^tm1Unc) mice and litter mate controls. Mice were >6 generations on the C57Bl/6 background. Mice were irradiated and given bone marrow by retro-orbital administration. None of our animals died using this protocol suggesting efficiency of engraftment. CF mice given CF bone marrow showed the least ability to survive, averaging around 50% survival (black triangles, n = 12). Cftr ^tm1Unc null mice given wild type bone marrow (open triangles, n = 10) had 76% survival. WT mice given CF (closed circles, n = 12) had 80% survival. WT mice given WT bone marrow (open circles, n = 10) had 76% survival. WT mice infected without transplantation had 94 ± 6% survival compared to 50 ± 13% survival of CF mice infected without transplantation. Weight changes (B) are consistent survival (A).

FIGURE 3

Myeloid specific Cftr KO and KI and response to chronic infection. (A) Schematic of the Cftr ^fl10 or Cftr ^invfl10 alleles with and without Cre recombinase present. Primers P1-P4 were used to detect the different alleles (specifics in the methods section). The Cftr ^fl10 conversion to the KO allele is a one way reaction whereas the Cftr ^invfl10 conversion to the KI allele is bidirectional. (B) DNA amplification of the region surrounding exon 10 from various tissues of mice homozygous for Cftr ^fl10 or Cftr ^invfl10 with and without LysMCre. Mice carrying the Cftr ^fl10 allele display no deletion of exon 10 (408 bp) but with LysMCre display at least some of the deleted product KI (148 bp) in bone marrow derived macrophages (M), bone marrow (BM), BAL cells (B), lung (Lu), kidney (Ki) and Liver (Li). Mice carrying the Cftr ^invfll10 allele display the inverted exon 10 (563 bp) but with LysMCre display inversion of at least some of the KI allele (408 b+p) (C,D) Mice were infected with P. aeruginosa and followed for 10 days. Myeloid specific KI (n = 6) and KO (n = 6) models were compared with the WT control (n = 5) mice and each other for BAL neutrophil numbers (C) and P. aeruginosa CFUs (D). The KO had significantly elevated neutrophils (p ≤ 0.05) and more bacteria (p ≤ 0.05) in the BAL than the WT control, whereas the KI model had comparable levels of neutrophils and bacteria. The KI levels of neutrophils and CFUs were significantly less than the KO model (p ≤ 0.05).

FIGURE 4

Exogenous WT BMDM decrease lung inflammation and infection in vivo. CF mice (n = 12) and WT controls (n = 10) were chronically infected with P. aeruginosa and followed for up to 10 days. Mice were infused with 10⁶ WT BMDM at day 1 post-infection. Animals were euthanized and evaluated for (A) white cell count, including a decrease in neutrophils (insert), (B) gross lung pathology and (C) P. aeruginosa infection burden. Treatment with bone marrow derived macrophages resulted in significantly (*) decreased recruitment of white blood cells improved gross lung pathology score and bacterial burden (p < 0.05).

FIGURE 5

hMSCs improve BMDM Inflammatory Response to Pathogens. CF BMDM (n = 3) preparations and WT BMDM (n = 2) treated with LPS demonstrated IL-6 and TNFα secretion. This was used as to explore the suppressive capacity of three different bone marrow donor hMSC supernatants. LPS stimulated CF and WT BMDM produce both IL-6 and TNFα. Treatment of the different BMDM preparations with the different hMSCs donor preparations resulted in cumulative decreased IL-6 (p ≤ 0.05) and TNFα (p ≤ 0.05) regardless of whether the BMDM were derived from CF (A,B) or WT mice (C,D). This is consistent with previously published data (Leyendecker et al., 2018).

FIGURE 6

hMSC Effect on PPARγ and TNFα expression. BMDM from CF patients and CF mice were evaluated for PPARγ (A). Sputum was obtained from CF patients (n = 3) and compared to healthy control (n = 3), demonstrating deficient PPARγ (p ≤ 0.05). BMDM from Cftr deficient mice also had deficient expression of PPARγ (p ≤ 0.05). (B) BMDM were stimulated with LPS and processed for PPARγ and TNFα gene expression demonstrating a decreased of PPARγ (p ≤ 0.05) and increased TNFα (p ≤ 0.05) relative to the unstiluated control of PPARγ and TNFα (2.1 ± 0.07 dCT and 1.7 ± 1.0 dCt respectively, n = 3) at baseline. The same culture conditions were done in the presence of hMSC condition medium. hMSC supernatants increased PPARγ and decreased TNFα (p ≤ 0.05) above the baseline controls.

FIGURE 7

Immune supportive therapy model for cystic fibrosis. Patients with CF early become infected with pathogens which contributed to the triad of infection → inflammation → lung-damage. The lung damage continues to create a pulmonary milieu that is susceptible to infection, so the triad continues resulting in the vicious circle of that is pathologic in CF. The feasibility of providing immune support focusing on hMSCs and BMDM which could be harnessed to skew the balance of the host response to infection and establishing chronic inflammation. Macrophages and hMSCs both have their potential roles in providing clinical efficacy and potency, but the key is likely how they interact in vivo. Macrophages defining the hMSC phenotype due to the milieu elicited and the contribution of the functionally tissue cued hMSCs to support the resolution toward homeostasis and tissue recovery. The hematopoietic approach would minimize the CFTR induced damage to the lung and other tissue, while at the same time promoting the patient’s management of their internal milieu. CRISPR/Cas9, the advent of iPSC cells and other gene editing technologies opens the door toward the potential adding corrective immune support in CF.