• UIP/IPF Usual interstitial pneumonia/ idiopathic pulmonary fibrosis
• AE-IPF Acute exacerbation of idiopathic pulmonary fibrosis
• BALF Bronchoalveolar lavage fluid
• NE Neutrophil Elastase
• CTX cotrimoxazole
• MFI mean fluorescent intensity
• FPR  Formyl peptide receptor
• ROS Reactive oxygen series
• PMA Phorbol 12-myristate 13-acetate
• f-MLP N-formyl-Met-Leu-Phe
• E Coli Escherichia Coli
• TGF-β Transforming growth factor beta

The standardised Phagoburst kit was used for the stimulation of leucocytes with quantitative measurement of leucocyte oxidative burst following 3 different stimulants: - E coli, f-MLP and PMA using flow cytometry.

Idiopathic pulmonary fibrosis (IPF) is characterised by extracellular matrix deposition from an unknown insult to the lung epithelium leading to the recruitment of myofibroblasts with dysregulated repair. The exact contribution of the immune system to this process is unclear, but neutrophils and macrophages play a part. Historically IPF was treated as an auto-immune process, but immune suppression was shown to have detrimental effects [1]. Acute IPF exacerbations (AE-IPF) carry a mortality of >50% which has important implications for this disease; since 4-20% of patients have an AE-IPF event each year [2-4]; and survivors show a shortened life expectancy with likely oxygen dependence. Neutrophils are frontline in host defence and excessive neutrophil activation and degranulation can cause lung injury from Reactive Oxygen Series (ROS) and released proteases [5]. In IPF the percentage of neutrophils in bronchoalveolar lavage fluid (BALF) at presentation is an independent predictor of mortality, with the doubling of neutrophil percentage increasing mortality by 30% regardless of age, lung function or smoking status [6].  Survival has been shown to be best in those with BALF neutrophils <3%, suggesting that neutrophil accumulation in the alveolar space induces injury and has a role in acute exacerbations and lung fibrosis [6]. The subsequent hypoxia would then amplify the inflammation and neutrophil degranulation but reduce anti-microbial protection [5]. Inflammatory signals from ‘formyl peptides’ released by bacteria or apoptotic cells and dying mitochondria will stimulate the formyl peptide receptors (FPR’s) increasing the release of neutrophil proteases including ‘neutrophil elastase’ leading to activation of the fibrotic cytokine transforming growth factor-β (TGF-β) [7-9]  Recent data shows that bacteria in the lower airway are associated with IPF progression, forced vital capacity decline and mortality in the first year which is independent of age, sex and lung function at diagnosis [10].  Formerly, the lower airway was considered to be sterile, but studies of BALF from new IPF patients show a 2-fold higher bacterial load than healthy controls [3,11] increasing to 4-fold with exacerbations.  Culture independent techniques using16-bacterial ribosomal RNA analysis have confirmed this and shown Haemophilus Influenzae, Streptococcus and Staphylococcus species to be more frequent [11-13]. It is unclear if the increased bacterial load is the cause or the consequence of the acute exacerbation [14]. However, germ free mice with pulmonary fibrosis are protected from increased mortality raising interest in antibacterial therapy [13]. Furthermore, fibrotic tissue creates a “salty microenvironment” that facilitates bacterial growth. Interestingly, corisin, a peptide conserved in Staphylococcus species has been shown to induce apoptosis of murine epithelial cells after intra pulmonary instillation or after exposure to corisin containing bacteria. Lung Corisin levels are raised in human AE-IPF and stimulate monocyte and neutrophil infiltration and collagen deposition in the lungs [15-17].  Additionally, Staphylococcus Aureus can secrete a Formyl Peptide Receptor-1 inhibitory protein for immune system evasion but the importance of this in AE-IPF is currently unknown[11]. There is now increasing evidence that IPF is associated with neutrophil accumulation, bacterial infection, increased oxidative stress and high levels of ROS leading to activation of the fibrotic cytokine TGF-β [18- 20].  Certainly, neutrophil depletion is shown to be protective against bleomycin induced lung injury [21]. Formyl Peptide Receptors have recently been shown to be tissue specific drivers of pulmonary fibrosis, with FPR-1 deficient mice protected from bleomycin-induced pulmonary fibrosis compared with wild-type mice [7-23]. Neutrophil recruitment to the lungs following bleomycin was reduced in FPR-1 deficient mice with adoptive transfer experiments showing this to be intrinsic to the absence of the FPR-1 [7,24]. Cotrimoxazole (trimethoprim and sulphamethoxazole) is very effective against intracellular Staphylococcus Aureus with excellent cell penetration of the human neutrophil and intracellular killing [25- 27]. Several clinical studies have demonstrated its protection against AE-IPF but the likely mechanism of this effect has not yet been explained [28-31].  A recent Nationwide analysis of Japanese IPF patients ventilated for rapidly progressive respiratory failure, showed a significant benefit in survival if  cotrimoxazole was given along with rescue steroids Odds ratio 0.2 (p=0.001)[31]. The authors suggested the benefit may relate to antimicrobial effects against Pneumocystis Jiroveci as well as anti-inflammatory effects on neutrophil-derived oxidative stress [31, 32]. Many antibiotics have effects on immune function. Studies of the sulphonamide dapsone (4, 4-diaminodiphenol sulphone), which shares the same sulphone ring as sulphamethoxazole, confirmed immunological effects on neutrophils [33- 37]. Dapsone was shown to reduce the generation of oxygen free radicals by neutrophils in a dose dependant manner. This inhibition was via dapsone’s ability to block the surface formyl peptide receptors (FPR) on neutrophils and hence block the release of both intracellular and extracellular ‘ROS’ [33-39]. Blocking of the FPR has been shown to reduce endothelial damage, lipid peroxidation and apoptosis induced by reactive neutrophils [34]. Formyl peptide receptors are transmembrane G-protein coupled receptors critical in myeloid trafficking for infection, inflammation and the immune response. They are present on monocytes, but no formal monocyte studies have been performed for dapsone. Dapsone was shown to reduce intracellular Protein Kinase-C activation by PMA (Phorbol 12-myristate 13-acetate); thereby reducing, but not completely blocking, the generation of intracellular oxidants that could further activate neutrophils [39, 40]. Few studies for cotrimoxazole exist apart from Anderson, who showed that cotrimoxazole reduced neutrophil-derived oxidative stress both in vivo and vitro but again monocyte effects were not examined [41]. This data evaluated the effects of cotrimoxazole upon neutrophil and monocyte activation in IPF patients in our long-term cotrimoxazole study along with the neutrophil and monocyte responses of healthy medical staff, untreated IPF patients and healthy controls. Neutrophils and monocytes were stimulated with E coli bacteria, Phorbol 12-myristate 13-acetate (PMA) and the bacterial peptide N-formyl-Met-Leu-Phe (f-MLP). The effects were assessed using a standardised Phagoburst kit with flow cytometric analysis. The study was approved by the National Research Ethics Committee for London-South East (REC reference 2006-004927-12). The ISTCRN registration number is 87032740.

Patients and healthy controls:-30 IPF patients on long-term treatment with cotrimoxazole (960mgBD).

24 Untreated IPF patients.

33 Healthy controls.

9 Healthy medical staff at baseline and pre and post cotrimoxazole for 7 days (960mgBD). 

9 Healthy medical staff at baseline and post trimethoprim for 7 days (200mgBD).

IPF Patients on Long-Term Cotrimoxazole: A total 30 patients with usual interstitial pneumonia /idiopathic pulmonary fibrosis (UIP/IPF) were examined. None of these patients were taking prednisolone, other immunosuppressant drugs nor anti-fibrotic drugs. All were clinically stable without intercurrent infections or current exacerbations. The dose of cotrimoxazole was 960mg BD as per the clinical trial and treatment had been ongoing for a mean of 38 months (range3-72months). Mean age 72.

Untreated Newly Diagnosed IPF: New IPF Patients Had Their Neutrophils And Monocytes Studied Prior To Commencing The Study.  These 24 Patients Were Stable Without Exacerbations Or Any Other Antibiotics Treatments And Were Not Taking Steroids, Anti-Fibrotics Or Other Treatments That Could Affect Their Neutrophil And Monocyte Function. Mean Age73

Healthy controls (HC): 33 healthy subjects without respiratory or autoimmune diseases formed the control group. None of whom were taking antibiotics, steroids or other immunosuppressive drugs that could affect neutrophil or monocyte function. Mean age 59.

9 Healthy Medical Staff (MS): Medical staff were healthy without intercurrent infections, other antibiotics therapy or treatments that could affect their Immune function. They were studied pre- and at day 7 of cotrimoxazole (CTX) (960mg BD) and later pre- and at day 7 of trimethoprim (200mg BD). A wash-out period of at least 2 months occurred between CTX and Trimethoprim. Mean age 50.

Blood samples: Blood was freshly drawn for the study along with an EDTA sample taken for a full blood count and differential.   4 mls of heparinised whole blood was used for the analysis of the oxidative burst tests of the neutrophils and monocytes. The blood was kept at room temperature with analysis commenced for all subjects within 2 hours of collection.  The blood was used whole and not subjected to any procedures that could modify the phagocyte function.

Neutrophil and Monocyte oxidative burst test: Flow cytometric methods and the standardised Phagoburst Kit (Glycotope biotechnology, Germany) [42] were used to measure rapid intracellular oxidative reactions within single cells to various stimulants.  This is an established method in the assessment of neutrophil and monocyte responses to detect abnormalities in their immune responses. The phagoburst analysis allows for quantitative determination of leucocyte oxidative burst in heparinised whole blood. Unlabelled opsonized E Coli bacteria are the particulate stimulant, and the protein kinase-C ligand Phorbol 12-myristate 13-acetate (PMA) as a high stimulus and an inducer of oxidative stress.  The bacterial peptide N-formyl-Met-Leu-Phe (f-MLP) is used as a physiological stimulant to activate the phagocyte formyl peptide receptors (FPR) generating Reactive Oxygen Series (ROS) much of which is extracellular.

Measurement of Oxidative Burst with Phagoburst Kit: Upon stimulation, granulocytes and monocytes produce reactive oxygen metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid by membrane bound NADPH oxidase) which function to destroy bacteria. The Phagoburst kit uses dihydrorhodamine (DHR-123) as a fluorogenic substrate to determine the percentage of reactive oxidants formed during the oxidative burst via the conversion of DHR-123 to R-123. This allows the percentage of phagocytic cells that produce reactive oxidants and their enzymatic activity (the amount of R-123 per cell) to be determined. The reaction is stopped by the addition of lysing agents which removes red cells and gives partial fixation of leukocytes. The percentage of cells that produce reactive oxygen radicals can then be analysed along with their mean fluorescent intensity.

100µl of whole blood was separately incubated at 37^oC with 20 µl of each of the following agents

Opsonized unlabelled E Coli (1-2X10⁹ bacteria/ ml) used undiluted, 

PMA (1. 62 mM stock) diluted 1 in 200

f-MLP (1mM stock) diluted 1 in 20

Reagent buffer (Instamed-salts) used as the negative background control for each stimulus. 

The 37^oC incubation time was 10 minutes for E Coli and PMA and reagent buffer but reduced to 2 minutes for f-MLP in order to capture its peak oxidative burst parameters as shown in several studies [43]. After this initial incubation period, 5 µl of CD14 (a monocyte marker) was added to each tube along with the DHR123 substrate and incubated at 37^oc   for a further 10 minutes. Following this, lysis of red cells by the addition of 2mls of kit lysing agent (diluted 1 in 10 with distilled water) was then commenced and left at room temperature for 20minutes, followed by centrifugation at 1500rpm (250g) at 2-8^oc  for 5 minutes.

Following this initial lysis, 3 washes with further centrifugation and decanting was undertaken (using 3 mls of wash buffer per tube each time) with the same centrifuge settings. Finally 1 ml of buffer was added to each tube for flow cytometric analysis. The tubes were protected from light and read within 30minutes.

Flow Cytometric analysis used a blue-green light (488nm Argon-ion laser) and 100, 000 leucocytes were analysed per tube. The percentage of cells producing reactive oxygen metabolites (%-recruitment) were analysed as well as their mean fluorescence intensity (MFI) with the relevant leucocyte clusters gated by the software in scatter diagrams. Monocytes were gated and visible by their green fluorescence (CD14 monocyte marker) and neutrophil were red on the dot plot forward and side scatter gating plots used to identify the neutrophils and monocytes populations. Fluorescent emissions from 10 000 cells per sample were recorded on a log scale. Respiratory burst was expressed as a % activated phagocytes (%-recruitment) and their cell activation intensity known as mean fluorescence intensity (MFI).

Calculation of mean fluorescence and Percent of recruited phagocytes

Basal (background buffer) mean fluorescent intensity (MFI) was subtracted from each stimulus (Ecoli, PMA and fMLP) to correct for spontaneous basal activity of the monocytes and neutrophils.  From this data, the mean oxidative burst of the neutrophils and monocytes could be measured as their mean fluorescent intensity (MFI) and compared with healthy controls and untreated IPF and other treatment groups shown in table 1+2. Also the percentage of activated (recruited) cells showing reactive oxygen generation responses (%-recruited phagocytes) was automatically recorded as a % by the flow cytometer shown in figure 1+2. The expected minimal normal ranges for MFI and %-recruitment for neutrophils and monocytes are given on (Table 1 and Figure 1)

Figure 1: Percent stimulation of peripheral blood Neutrophil + Monocyte with PMA, fMLP and E Coli for Healthy control, cotrimoxazole (CTX) treated and untreated IPF patients.

Table 1:  Neutrophils and monocytes responses in IPF patients and healthy controls.

Neutrophils Mean fluorescence Intensity* ±SEM (arbitrary units)	Healthy controls (HC) Mean ± SEM N=33	Untreated IPF N=24	IPF on long-term CTX (mean 38 months) N=30	Mann Whitney U test HC versus untreated IPF	Mann Whitney U test HC versus IPF on CTX	Mann Whitney U test Untreated IPF versus IPF on CTX
Mean neutrophil blood count	3.63 ± 0.20	5.08± 0.29	4.31± 0.27	P=0.0003*	P=0.025*	P=0.05
PMA N	7718± 772	7212± 762	5962± 827	P=0.77	P=0.128	P=0.22
fMLP N	913 ± 152	778± 143	61± 96	P=0.70	P=0.0001+	P=0.0001+
E coli N	3892 ± 406	3506± 529	4264±599	P=0.43	P=0.88	P=0.454
Monocytes Mean fluorescence Intensity * ±SEM
Mean monocyte blood count 109 /l ±SEM	0.53± 0.03	0.74± 0.17	0.69±0.03	P=0.0001*	P=0.0001*	P=0.11
PMA M	1490 ± 201	1485± 311	787± 123	P=0.43	P=0.016+	P=0.016+
fMLP M	1075± 203	870± 214	124± 94	P=0.194	P=0.0001+	P=0.0004+
E coli M	918± 134	755± 124	519± 98	P=0.613	P=0.02	P=0.12
Mean Fluorescent intensity minimum normal values in healthy controls	E Coli	PMA	F-MLP
Neutrophils	397-1169	574-2594	276-606	CTX-cotrimoxazole TMP-trimethoprim + Significance
Monocytes	304-693	339-895	241-515

The Prism3. 03 and sigma stats 3. 1 software packages were used for all statistical analyses. Baseline data for each subject, group and stimulus were calculated including peripheral blood neutrophil and monocyte counts, and results for mean fluorescence intensity (MFI) and the percentage of recruited cells (%-recruited) following the 3 stimulants. Column statistics were used to determine means and standard error of the mean (SEM). Statistical comparison between unmatched groups (MS, HC, treated and untreated IPF), were made by 2-tailed Mann-Whitney U tests for non-parametric data. Significance was initially set at the 5% level. However, to allow for multiple comparisons, the Bonferroni correction was invoked and the significance was then set at <0. 0166 (0. 05÷3) for the healthy controls and treated and untreated IPF group as indicated on the table 1. The Medical staff data (pre and post trimethoprim and cotrimoxazole) were analysed by paired T test with significance at <0. 05 as indicated on table 2 and figure 2.

Figure 2: Healthy Medical staff results of neutrophil monocyte% stimulatio n with PMA, FMLP and E Coli.

The Healthy Controls (HC)

The 33 healthy controls showed normal neutrophil and monocyte blood counts (Table 1). Stimulation of their neutrophils and monocytes fell in the normal range for %-recruitment and MFI to PMA, f-MLP and Ecoli (Table 1+ figure 1).  Opsonized Ecoli and PMA are more powerful stimulants to phagocytes and generated >95% of neutrophil recruitment with monocytes recruitment lower at 80% activation (Figure 1). f-MLP is a physiological bacterial peptide stimulant and gives much lower levels of %-recruitment via the surface formyl peptide receptors. Healthy controls showed a mean 10% stimulation of both cell types with f-MLP (Figure 1).

Untreated IPF Patients

These patients had significantly higher peripheral neutrophil and monocyte counts compared with healthy controls (p=0. 0003 +p=0. 0001) respectively. Stimulation of their neutrophils and monocytes did not produce any significant differences in MFI or %-cell recruitment compared with the healthy controls to the 3 stimulants (PMA, Ecoli + f-MLP). Table 1 + Figure 1.

Treated IPF Patients with Cotrimoxazole (CTX) For a Mean Duration of 38months

For Ecoli, there was no significant difference in responses of the neutrophils and monocytes compared with healthy controls and untreated-IPF groups. Neutrophil stimulation with PMA showed a non-significant reduction in the %-recruited cells (figure 1) and MFI compared with HC and untreated IPF.  PMA stimulation of the monocytes showed a 48% reduction in MFI compared with HC (p=0. 016) despite the higher monocyte count, but %-recruited cells was unchanged. MFI following f-MLP stimulation was significantly reduced for neutrophils (96%) and monocytes (84%) compared with both HC and untreated-IPF, while %-recruitment was minimally affected (figure 1). These findings indicate significant blockade of the neutrophil and monocyte surface formyl peptide receptors to stimulation by f-MLP while taking CTX. This reduction in MFI is seen despite the higher monocyte counts relative to the HC group (Table1).

Peripheral Blood Counts In All IPF Patients

The 30 IPF patients on long-term CTX had statistically lower mean peripheral neutrophil counts (4. 31 Vs 5. 08 X 10⁹) compared with untreated IPF (p=0. 05). Blood monocyte counts were similar between treated and untreated IPF (0. 69 Vs 0. 74X10⁹: p=0. 11). Comparison to the healthy controls showed peripheral neutrophil count to be higher in treated IPF Vs HC (4. 31 Vs 3. 63 X109: p=0. 025) and likewise for monocytes (0.69 Vs 0.53X10⁹: p=0. 0001). Table 1.

Medical Staff (MS) Findings Before and After 7 Days of Cotrimoxazole (CTX) and Trimethoprim

The 9 MS showed normal stimulation of their neutrophils and monocytes to PMA, f-MLP and Ecoli immediately prior to either drug treatment with responses comparable to the larger healthy control group. The MS had normal peripheral neutrophil and monocyte counts [Table 2]. At day 7, MS on CTX, showed no changes in Ecoli phagocyte stimulation. PMA stimulation was reduced by 69% for monocytes only (p=0.033) similar to CTX-treated IPF patients. Stimulation by f-MLP was significantly reduced with CTX by 84% in neutrophils (p=0.0005) and 88% in monocytes (p=0.04) at day 7 for the MS [Table 2]. With trimethoprim, the MS showed no changes in E Coli or PMA stimulation. There was significantly reduced f-MLP stimulation of neutrophils (by 83%, p=0.01) and monocytes (by 40%, p=0. 014) table 2. The %-recruited neutrophils and monocytes at day 7 to f-MLP were reduced (Figure 2).

Results Summary: Our data shows normal oxidative burst function for both neutrophils and monocytes in the healthy controls, untreated IPF and medical staff pre-treatments. In treated IPF patients responses to f-MLP were significantly reduced by CTX due to blockade of their surface FPR’s in both neutrophil and monocyte. Monocyte responses to PMA were also significantly reduced, although like Dapsone not completely inhibited. Medical staff taking CTX for 7 days showed the same responses as the CTX-treated IPF cases indicating that the observations were specific to CTX and not modified by the IPF disease process. Trimethoprim also blocked stimulation by f-MLP significantly in neutrophils and monocytes with no significant effects on PMA responses. Both IPF groups (untreated and CTX) have statistically higher neutrophil and monocyte counts than controls. In this regard, a recent report has identified neutrophil counts >7. 5(x10⁶/l) and monocyte counts >0. 9(x10⁶/l), to both be associated with CT progression of the disease [44].

Table 2: Medical staff  responses to Cotrimoxazole and trimethoprim.

Neutrophils Mean fluorescence Intensity* ±SEM (arbitrary units)	Healthy Medical Staff N=9 Pre-CTX	Healthy Medical Staff N=9  post-CTX 7 days	Paired t test + significance <0.05 (pre- and post CTX)	Healthy Medical Staff N=9 Pre-TXP	Healthy Medical Staff N=9 post-TMP 7 days	Paired t Paired t test + significance <0.05 (pre-post TMP)
Mean neutrophil blood  count 109/l (SEM)	3.3± 0.20	3.7± 0.37	p=0.27	3.28± 0.33	3.54± 0.33	P=0.63
PMA N	6474± 1203	5902± 1260	P=0.7915	8534± 2076	7037± 2526	P=0.58
fMLP N	1577±  114	264± 524	P=0.0005+	1009± 386	281± 133	P=0.011+
E coli N	4529± 987	3261± 720	P=0.22	4529± 987	3261± 720	P=0.227
Monocytes Mean fluorescence Intensity * ±SEM	Healthy Medical Staff Pre- CTX	Healthy Medical Staff post-CTX	Paired t test + significance <0.05 ( pre- post CTX)	MS Pre-TMP	MS post-TMP  7 days	Paired t test + significance at <0.05 ( pre- post TMP)
Mean  monocyte blood count 109/l (SEM)	0.5± 0.06	0.5± 0.05	p=0.28	0.43±0.07	0.5± 0.03	P=0.22
PMA M	1262± 231	399± 148	P=0.033+	2926± 411	2078± 289	P=0.15
fMLP	1887±  758	161± 111	P=0.04+	918± 232	563± 152	P=0.014+
E coli	3362± 1785	1012± 168	P=0.22	1998± 888	1894± 289	P=0.91
	CTX-cotrimoxazole	TMP-trimethoprim

Our results suggest that cotrimoxazole and trimethoprim, like Dapsone, can significantly block the stimulation of the surface formyl peptide receptors on neutrophils and also monocytes. This will reduce the release of intra and extracellular ROS, helping to reduce recruitment and activation of neutrophils and monocytes in the alveolar space [7] and may offer protection in stable disease and even during exacerbations. Recruitment and oxidative burst is important for microbial protection but if excessive can cause endothelial damage. FPR-1 is the primary receptor for released bacterial Formyl Peptide’s and also Formyl Peptide’s released by dead and dying host cells and products of mitophagy [7,45]. As discussed, neutrophils play a role in alveolar damage and fibrosis in IPF with oxidation products, neutrophil elastase and TGF-β as important intermediaries. When Neutrophils migrate to inflamed tissue in response to released formyl peptide signals, the FPR-1 will be activated to deliver a high local concentration of ROS and proteolytic enzymes especially neutrophil elastase.[8,22,23,46] Locally increased cellular and oxidative stress contributes to alveolar epithelial cell injury and may induce their apoptosis given their reduced capacity to generate catalase (a free radical scavenger), leaving them prone to ROS injury[47,48]. Hypoxic neutrophils themselves have delayed apoptosis along with a 6-fold increase in neutrophil elastase release following stimulation by formyl peptides [49,50].  Dapsone has been shown to reduce neutrophil Elastase (NE) release by f-MLP [45]. NE may have a more central role in IPF lung injury than previously recognised as NE levels are raised in serum and BALF from IPF patients [8, 21,23,24,49]. The increased mortality seen with oral steroids in IPF, may relate to steroid-induced demargination of neutrophils and their delayed apoptosis facilitating increased ROS and NE release. The effect of delayed apoptosis upon the neutrophil bacterial load and corisin generation is unknown currently but future studies will hopefully clarify this. Three different formyl peptide receptors are recognised in man. FPR-1 is the most studied and abundantly expressed on neutrophils and monocytes and central to activation by infection or tissue damage [51].  FPR-2 is present mainly on neutrophils and epithelial cells and has lower affinity to f-MLP. It appears to bind lipid mediators such as lipoxin-A4 and amyloid protein-A along with annexin-A1. The latter is upregulated by steroids reducing neutrophil migration and apoptosis with stimulation their phagocytosis by monocytes [50,52].  FPR-3 is limited to eosinophils, monocytes and dendritic cells and the receptor is largely insensitive to formyl peptides and may be involved in allergic responses [51,52]. Murine studies show that intra-tracheal Bleomycin induces a neutrophilic alveolitis with NE release, inflammation and lung fibrosis, illustrating an important role for neutrophils and NE in fibrotic injury. Treatment of mice with the NE-inhibitor (Sivelestat) blocks bleomycin induced fibrosis, neutrophil and monocyte accumulation, epithelial cell detachment and activation of TGF β1 [53]. Intriguingly, NE is regulated by the endogenous protease inhibitor alpha-1 anti-trypsin (α1AT) which is a specific inactivator of NE and shown to prevent epithelial cell detachment in cell culture studies [8,19,21,50,54]. Blocking NE reduces TGFβ1 activation and neutrophilic alveolitis along with fibrosis. In NE deficient mice there is absent staining for active TGFβ1 in BALF and transbronchial lung biopsies, indicating that released NE has fibrogenic consequences [REF55]. Importantly, ROS activates TGFβ1 from its latent form which then drives fibroblast proliferation, matrix deposition and Epithelial Mesenchymal Transition (EMT) [20,52,56].  ROS increases sensitivity to growth factors and also TGFβ1 effects and produces endoplasmic reticulum stress and apoptotic death in alveolar type-2 epithelial cells [57]. Collectively this promotes myofibroblast differentiation in IPF, and myofibroblasts are shown to be more resistant to ROS-induced apoptosis due to protective effects of TGFβ1as seen in cell culture studies [23,45,58]. Extracellular ROS drives myofibroblasts to release hydrogen peroxide thus stimulating further TGFβ1 release [47,59]. These processes show interdependence but a reduction in early activation of neutrophils and monocytes with reduced ROS and NE release may give therapeutic benefit through reduced TGF-β1 activation and EMT stimulation. Our previous studies of IPF patients taking long-term cotrimoxazole, showed a relative Forced Vital Capacity decline of -60ml/per year against and expected decline of -200 ml/year and reduced AE-IPF in those who continued treatment [30]. Reduced AE-IPF were noted also in the TIPAC study [29]. The recently published EME-TIPAC study of IPF patients taking anti-fibrotic drugs (nintedanib or pirfenidone)  did not show added benefit from CTX in reducing death, exacerbations or hospital admission at 12 months in those patients already taking these drugs[60].  

The recent identification of the neutrophil formyl peptide receptor-1 as a tissue specific driver of pulmonary fibrosis may explain the association of neutrophils in alveolar lavage with disease progression, ROS generation and TGFβ1activation.  Our previous observations that IPF patients treated with CTX seldom develop acute exacerbations and show a reduced rate lung function decline may result from FPR-blockade by CTX as shown in this data. This central role of the neutrophils in IPF may open up new avenues for treatment and understanding of this condition.  

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